Taf1 inhibitors for the therapy of cancer

ABSTRACT

The present invention relates to lactam derivatives of formula (I) for use as medicaments as well as pharmaceutical compositions comprising these compounds, particularly for use as inhibitors of the bromodomain-containing protein TAF1 (i.e., transcription initiation factor TFIID subunit 1) and for use in the treatment or prevention of cancer.

The present invention relates to lactam derivatives of formula (I) foruse as medicaments as well as pharmaceutical compositions comprisingthese compounds, particularly for use as inhibitors of thebromodomain-containing protein TAF1 (i.e., transcription initiationfactor TFIID subunit 1) and for use in the treatment or prevention ofcancer.

Bromodomain proteins of the BET (bromodomain and extraterminal domain)family recognize histone lysine acetylation and mediate transcriptionalactivation of target genes such as the c-MYC oncogene. PharmacologicalBET domain inhibitors promise therapeutic benefits in a variety ofcancers. In particular, BRD4 (bromodomain-containing protein 4) is anacetyl-lysine reader of the BET family (Wang, R. et al., 2012; Dey etal., 2003; Filippakopoulos et al., Cell, 2012). This protein binds toacetylated histones at promoter and enhancer regions and recruitstranscription factors, cofactors and RNA polymerase II (RNApol II), thusmodulating the transcription of a subset of genes in a highly contextdependent way. Through its effect on target gene expression (Zuber etal., 2011; Wyce et al., 2013), the bromodomain-histone interaction playsa key role regulating cell cycle progression (Dey et al., 2003; Devaiahet al., 2013; Yang et al., 2008; Wu et al., 2007), genomic structure andstability (Wu et al., 2007; Floyd et al., 2013) and development ofseveral pathologies, including cancer (Zuber et al., 2011; Yang et al.,2008; Nagarajan et al., 2014; Wu et al., 2015). The design of chemicalprobe compounds targeting the two bromodomains of BRD4, such as thepan-BET inhibitors JQ1 (Filippakopoulos et al., 2010) and I-BET-151(Seal et al., 2012) and their compelling efficacy in cancer models, hasprompted the development of drug candidates for these proteininteraction modules that are now undergoing clinical trials(Filippakopoulos et al., 2014).

Despite the large number of competing clinical programs, the mechanisticand chemical diversity of currently available BRD4 inhibitors is limited(Filippakopoulos et al., Cell, 2012; Filippakopoulos et al., 2014).Furthermore, there is a lack of detailed understanding of the factorsaffecting BRD4 function, and druggable targets upstream or downstream ofBRD4 have remained elusive.

In the context of the present invention, the inventors set out to designa strategy allowing the unbiased scouting of high diversity chemicalspace for modulators of a BRD4-dependent inactive chromatin state. Inthe background of the human haploid cell line KBM7 (Andersson et al.,1995), allowing unambiguous monoallelic genetic configurations, the RFP(Red Fluorescent Protein) gene was integrated in heterochromatic lociwhich are specifically activated by BRD4 inhibition. As described inExample 1, a high-diverse compound library of 89,355 small molecules wasthen chosen and compounds were selected for their ability to reactivateRFP expression. The efficient identification of many BRD4 inhibitors,including all the BET inhibitors in this library, validated theexperimental strategy. Importantly, the setup allowed the identificationof small molecules that efficiently induced RFP expression but failed tobind BRD4, indicating a novel mechanism of action that mimics BRD4inhibition without direct engagement. The inventors surprisingly foundthat one such compound, CeMMEC1, functioned by binding and potentlyinhibiting the second bromodomain of the transcription initiation factorTAF1. Moreover, by investigating the properties of this new compound andits derivatives, the inventors surprisingly found a strong synergybetween the targeting of TAF1 and BRD4, which resulted in efficientkilling of BRD4-dependent cancer cells, as also described in Example 1.

TAF1 is the largest component of the TAF subunits contained in the TFIIDcore, which is part of the pre-initiation complex (PIC) and serves torecognize the TATA box and correctly place RNAPol II for transcriptioninitiation (Lee et al., 2005; Kloet et al., 2012; Kandiah et al., 2014).Thereby, TAF1 plays a fundamental role in the assembly of thetranscription machinery. Similar to BRD4, TAF1 is essential for theviability of many different cell lines (Wang et al., 2015; Blomen etal., 2015), and the two proteins interact not only in the regulation oftranscription but also physically in co-immunoprecipitation experiments.It has been demonstrated in the context of the present invention thatTAF1 knockdown increases sensitivity to BRD4 inhibition, and BRD4inhibitors synergize with TAF1 inhibitors (such as the compounds offormula (I) provided herein) to impair viability of BRD4-dependentcancer cell lines. Thus, while the specific functions of thebromodomains of TAF1 have remained elusive, the results provided hereinindicate that the second bromodomain of TAF1 is a relevant target inBRD4 driven cancers. Certain BRD4 inhibitors, including bromosporine anda specific 3,5-dimethylisoxazole derivative (McKeown et al., 2014), areknown to bind TAF1, but currently there is no specific inhibitoravailable for this bromodomain-containing protein.

The inventors also found a novel and potent, direct BRD4 inhibitor,i.e., CeMMEC2. Other bromodomain inhibitors have already been described,e.g., in WO 2012/174487, WO 2013/027168, WO 2014/076146, US2014/0135336, WO 2014/134583, WO 2014/191894, WO 2014/191896, US2014/0349990, WO 2014/191906, and WO 2016/016316. Furthermore, certainlactam derivatives have been disclosed as having other pharmacologicalactivities, e.g., in WO 2010/072597, WO 2013/068489, WO 2014/120808, WO2015/106272, and WO 2016/004417.

The present invention solves the problem of providing novel potentinhibitors of TAF1 which can advantageously be used in therapy,particularly in the treatment or prevention of cancer. The inventionalso provides TAF1 inhibitors that are highly selective for TAF1 overother bromodomain-containing proteins, particularly BRD4.

Accordingly, the present invention provides a compound of the followingformula (I)

or a pharmaceutically acceptable salt, solvate or prodrug thereof, foruse as a medicament and, in particular, for use in the treatment orprevention of cancer.

In formula (I), ring B is a group having the following structure:

In ring B, one of the ring atoms X₂ and X₃ is N(R^(X1)), and the otherone of said ring atoms X₂ and X₃ is C(═O).

The ring atom X₁ is selected from N(R^(X1)), C(R^(X2)) and C(═O), andthe ring atoms X₄ and X₅ are each independently selected from N(R^(X1)),C(R^(X3)) and C(═O), wherein at least one of said ring atoms X₁, X₄, andX₅ is different from N(R^(X1)) and C(═O).

In the compounds of formula (I), if X₃ and X₅ are C(═O), X₄ isN(R^(X1)), and X₁ is C(R^(X2)), then X₂ is N(H).

Each

is independently a single bond or a double bond, wherein at least one ofany two adjacent bonds

is a single bond.

Each R^(X1) is independently selected from hydrogen, C₁₋₅ alkyl,—CO(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-aryl, and —(C₀₋₃ alkylene)-heteroaryl,wherein the aryl comprised in said —(C₀₋₃ alkylene)-aryl and theheteroaryl comprised in said —(C₀₋₃ alkylene)-heteroaryl are eachoptionally substituted with one or more groups R^(X11).

R^(X2) is selected from hydrogen, C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅haloalkyl), —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃,—(C₀₋₃ alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl).

The two groups R^(X3) are either mutually linked to form, together withthe ring carbon atoms that they are attached to, a 5- or 6-memberedcyclyl group which is optionally substituted with one or more groupsR^(X31), or the two groups R^(X3) are each independently selected fromhydrogen, C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl),—O(C₁₋₅ alkylene)-OH, —O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅alkyl), —NH₂, —NH(C₁₋₅ alkyl), —N(C₁₋₅ alkyl)(C₁₋₅ alkyl), halogen, C₁₋₅haloalkyl, —O—(C₁₋₅ haloalkyl), —CF₃, —CN, —NO₂, —CHO, —CO—(C₁₋₅ alkyl),—COOH, —CO—O—(C₁₋₅ alkyl), —O—CO—(C₁₋₅ alkyl), —CO—NH₂, —CO—NH(C₁₋₅alkyl), —CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —NH—CO—(C₁₋₅ alkyl), —N(C₁₋₅alkyl)-CO—(C₁₋₅ alkyl), —SO₂—NH₂, —SO₂—NH(C₁₋₅ alkyl), —SO₂—N(C₁₋₅alkyl)(C₁₋₅ alkyl), —NH—SO₂—(C₁₋₅ alkyl), and —N(C₁₋₅ alkyl)-SO₂—(C₁₋₅alkyl).

Each R^(X11) is independently selected from C₁₋₅ alkyl, C₂₋₅ alkenyl,C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl),—(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen,alkylene)-(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl),alkylene)-CF₃, —(C₀₋₃ alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃alkylene)-CHO, —(C₀₋₃ alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH,—(C₀₋₃ alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl),—(C₀₋₃ alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl).

Each R^(X31) is independently selected from C₁₋₅ alkyl, C₂₋₅ alkenyl,C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl),—(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃alkylene)-(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃alkylene)-CF₃, —(C₀₋₃ alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃alkylene)-CHO, —(C₀₋₃ alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH,—(C₀₋₃ alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl),—(C₀₋₃ alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl).

Ring B is attached to the remainder of the compound of formula (I) viathe ring carbon atom that is marked with an asterisk (*) or, if X₄ andX₅ are each C(R^(X3)) and the two groups R^(X3) are mutually linked toform, together with the ring carbon atoms that they are attached to, a5- or 6-membered cyclyl group which is optionally substituted with oneor more groups R^(X31), then ring B may also be attached to theremainder of the compound of formula (I) via any ring carbon atom ofsaid 5- or 6-membered cyclyl group.

Ring A is aryl or heteroaryl, wherein said aryl and said heteroaryl areeach optionally substituted with one or more groups R^(A).

Each R^(A) is independently selected from C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅haloalkyl), —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), alkylene)-CF₃, —(C₀₋₃alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-cycloalkyl, —(C₀₋₃ alkylene)-O-cycloalkyl, —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-cycloalkyl, —(C₀₋₃alkylene)-heterocycloalkyl, —(C₀₋₃ alkylene)-O-heterocycloalkyl, and—(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-heterocycloalkyl.

L is selected from —CO—N(R^(L1))—, —N(R^(L1))—CO—, —CO—O—, —O—CO—,—C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—, —C(═S)—N(R^(L1))—,—N(R^(L1))—C(═S)—, —N(R^(L1))—CO—N(R^(L1))—, —N(R^(L1))—CO—O—,—N(R^(L1))—C(═N—R^(L2))—N(R^(L1))—, —O—C(═N—R^(L2))—N(R^(L1))—,—N(R^(L1))—C(═N—R^(L2))—O—, —S—C(═N—R^(L2))—N(R^(L1))—,—N(R^(L1))—C(═S)—N(R^(L1))—, —O—C(═S)—N(R^(L1))—, —N(R^(L1))—C(═S)—O—,—S—CO—N(R^(L1))—, and —N(R^(L1))—CO—S—.

Each R^(L1) is independently selected from hydrogen and C₁₋₅ alkyl.

Each R^(L2) is independently selected from hydrogen, C₁₋₅ alkyl, —CN,and —NO₂.

n is 0 or 1.

m is 0 or 1,

The present invention also provides a pharmaceutical compositioncomprising a compound of formula (I), as described and defined herein,or a pharmaceutically acceptable salt, solvate or prodrug thereof, incombination with a pharmaceutically acceptable excipient.

Moreover, the invention relates to the use of a compound of formula (I)or a pharmaceutically acceptable salt, solvate or prodrug thereof in thepreparation of a medicament, particularly for the treatment orprevention of a disease/disorder such as, e.g., cancer.

The compounds of formula (I) have been found to be potent inhibitors ofTAF1, specifically of the second bromodomain of TAF1, as alsodemonstrated in the appended examples, and can thus be used for thetreatment or prevention of cancer, particularly BRD4-driven cancerand/or c-MYC-driven cancer, as well as other diseases/disordersassociated with TAF1 and/or BRD4.

The present invention thus particularly relates to a compound of formula(I), as described and defined herein, or a pharmaceutically acceptablesalt, solvate or prodrug thereof, for use in the treatment or preventionof cancer.

The invention also provides a pharmaceutical composition comprising acompound of formula (I) or a pharmaceutically acceptable salt, solvateor prodrug thereof, in combination with a pharmaceutically acceptableexcipient, for use in the treatment or prevention of cancer.

The present invention furthermore relates to the use of a compound offormula (I) or a pharmaceutically acceptable salt, solvate or prodrugthereof in the preparation of a medicament for the treatment orprevention of cancer.

The invention likewise provides a method of treating or preventingcancer, the method comprising administering a compound of formula (I) ora pharmaceutically acceptable salt, solvate or prodrug thereof, or apharmaceutical composition comprising any of the aforementioned entitiesand a pharmaceutically acceptable excipient, to a subject (e.g., ahuman) in need thereof.

Moreover, the present invention relates to a compound of formula (I) ora pharmaceutically acceptable salt, solvate or prodrug thereof, or apharmaceutical composition comprising any of the aforementioned entitiesand a pharmaceutically acceptable excipient, for use in inhibiting TAF1or for use in treating or preventing cancer by inhibiting TAF1. Theinvention further refers to the use of a compound of formula (I) or apharmaceutically acceptable salt, solvate or prodrug thereof in thepreparation of a medicament for inhibiting TAF1 or for treating orpreventing cancer by inhibiting TAF1. In addition thereto, the inventionprovides a method of inhibiting TAF1 in a subject, the method comprisingadministering a compound of formula (I) or a pharmaceutically acceptablesalt, solvate or prodrug thereof, or a pharmaceutical compositioncomprising any of the aforementioned entities and a pharmaceuticallyacceptable excipient, to a subject (e.g., a human) in need thereof. Theinvention also provides a method of treating or preventing cancer byinhibiting TAF1, the method comprising administering a compound offormula (I) or a pharmaceutically acceptable salt, solvate or prodrugthereof, or a pharmaceutical composition comprising any of theaforementioned entities and a pharmaceutically acceptable excipient, toa subject (e.g., a human) in need thereof.

The invention further provides novel compounds embraced by formula (I),particularly the compounds 1, 3, 4, 5, 6, 8, 10, 12, 13, 15, 16, 24, 25,26, 27, 29, 30, 33, 36, 37, 38 and 39 (as shown further below) as wellas pharmaceutically acceptable salts, solvates and prodrugs of any ofthese compounds.

The present invention also relates to a TAF1 inhibitor (which ispreferably a compound of formula (I) or a pharmaceutically acceptablesalt, solvate or prodrug thereof) for use in therapy, particularly foruse in the treatment or prevention of cancer, wherein the TAF1 inhibitoris to be administered in combination with a BRD4 inhibitor. Theinvention likewise relates to a BRD4 inhibitor for use in therapy,particularly for use in the treatment or prevention of cancer, whereinthe BRD4 inhibitor is to be administered in combination with a TAF1inhibitor. Moreover, the invention provides a pharmaceutical compositioncomprising a TAF1 inhibitor and a BRD4 inhibitor, and its use intherapy, particularly for use in the treatment or prevention of cancer.The invention further provides a method of treating or preventingcancer, the method comprising administering a TAF1 inhibitor incombination with a BRD4 inhibitor to a subject (e.g., a human) in needthereof. The TAF1 inhibitor is preferably a compound of formula (I) or apharmaceutically acceptable salt, solvate or prodrug thereof, asdescribed and defined herein. The BRD4 inhibitor is preferably a directBRD4 inhibitor and may be, e.g., the compound CeMMEC2 shown below, or apharmaceutically acceptable salt, solvate or prodrug thereof, or any oneof the compounds JQ1 (also referred to as (S)-JQ1), I-BET 151 (orGSK1210151A), I-BET 762 (or GSK525762), PF-1, bromosporine, OTX-015,TEN-010, CPI-203, CPI-0610, RVX-208, B12536, TG101348, LY294002, or apharmaceutically acceptable salt, solvate or prodrug of any of theseagents, or any one of the compounds disclosed in WO 2012/174487, WO2014/076146, US 2014/0135336, WO 2014/134583, WO 2014/191894, WO2014/191896, US 2014/0349990, or WO 2014/191906.

The invention furthermore relates to the compound CeMMEC2 or apharmaceutically acceptable salt, solvate or prodrug thereof, or apharmaceutical composition comprising any of the aforementioned entitiesand a pharmaceutically acceptable excipient, for use in therapy,particularly for use in the treatment or prevention of cancer (includingany one of the specific types of cancer referred to herein). Theinvention also refers to each one of the compounds depicted in FIG. 11,particularly CeMMEC3, CeMMEC4, CeMMEC5, CeMMEC6, CeMMEC7, CeMMEC8,CeMMEC9, CeMMEC10, CeMMEC11, CeMMEC12, or a pharmaceutically acceptablesalt, solvate or prodrug of any of these compounds, or a pharmaceuticalcomposition comprising any of the aforementioned entities and apharmaceutically acceptable excipient, for use in therapy, particularlyas functional BRD4 inhibitors, and particularly for use in the treatmentor prevention of cancer.

The present invention relates to the treatment or prevention of cancer,particularly BRD4-dependent cancer and/or c-MYC-dependent cancer, usinga compound of formula (I) as described and defined herein, optionally incombination with a BRD4 inhibitor (such as, e.g., CeMMEC2). The cancerto be treated or prevented in accordance with the invention ispreferably selected from prostate carcinoma, breast cancer, acutemyeloid leukemia, multiple myeloma, glioblastoma, and NUT midlinecarcinoma.

The present invention furthermore relates to the use of a compound offormula (I) or a pharmaceutically acceptable salt, solvate or prodrugthereof as a TAF1 inhibitor in research, particularly as a research toolcompound. Accordingly, the invention refers to the in vitro use of acompound of formula (I) or a pharmaceutically acceptable salt, solvateor prodrug thereof as a TAF1 inhibitor and, in particular, to the invitro use of a compound of formula (I) or a pharmaceutically acceptablesalt, solvate or prodrug thereof as a research tool compound acting as aTAF1 inhibitor. It is to be understood that the term “in vitro” is usedin this specific context in the sense of “outside a living human oranimal body”, which includes, in particular, experiments performed withcells, cellular or subcellular extracts, and/or biological molecules inan artificial environment such as an aqueous solution or a culturemedium which may be provided, e.g., in a flask, a test tube, a Petridish, a microtiter plate, etc. The invention likewise relates to an invitro method of inhibiting TAF1, comprising the use of a compound offormula (I) or a pharmaceutically acceptable salt, solvate or prodrugthereof as a TAF1 inhibitor. The present invention further provides amethod (particularly an in vitro method) of inhibiting TAF1 in a sample,the method comprising applying a compound of formula (I) or apharmaceutically acceptable salt, solvate or prodrug thereof to thesample.

The compound of formula (I) will be described in more detail in thefollowing.

In formula (I), ring B is a group having the following structure:

In ring B, one of the ring atoms X₂ and X₃ is N(R^(X1)), and the otherone of said ring atoms X₂ and X₃ is C(═O).

Preferably, X₂ is C(═O), and X₃ is N(R^(X1)).

The ring atom X₁ is selected from N(R^(X1)), C(R^(X2)) and C(═O), andthe ring atoms X₄ and X₅ are each independently selected from N(R^(X1)),C(R^(X3)) and C(═O), wherein at least one of said ring atoms X₁, X₄, andX₅ is different from N(R^(X1)) and C(═O). The requirement that at leastone of the ring atoms X₁, X₄, and X₅ is different from N(R^(X1)) andC(═O) can also be expressed as a requirement that at least one of thesering atoms must be C(R^(X2)) or C(R^(X3)).

Preferably, not more than one (if any) of the ring atoms X₁, X₄, and X₅is N(R^(X1)), and not more than one (if any) of said ring atoms X₁, X₄,and X₅ is C(═O). More preferably, the ring atom X₁ is C(R^(X2)), and thering atoms X₄ and X₅ are each C(R^(X3)).

In the compounds of formula (I), if X₃ and X₅ are C(═O), X₄ isN(R^(X1)), and X₁ is C(R^(X2)), then X₂ is N(H).

Each

is independently a single bond or a double bond, wherein at least one ofany two adjacent bonds

is a single bond (i.e., at least one of any two bonds that are attachedto the same ring atom is a single bond).

Preferably, at least one of the bonds

in formula (I) is a double bond. More preferably, the bond

between the ring atom X₁ and the ring carbon atom which is bound to themoiety —(CH₂)_(n)-L-(CH₂)_(m)— is a double bond, the bond

between said ring carbon atom which is bound to the moiety—(CH₂)_(n)-L-(CH₂)_(m)— and the ring atom X₅ is a single bond, and thebond

between the ring atoms X₄ and X₅ is a single bond or a double bond(preferably a double bond).

It is particularly preferred that the ring atom X₁ is C(R^(X2)), thering atoms X₄ and X₅ are each C(R^(X3)), the bond

between the ring atom X₁ and the ring carbon atom which is bound to themoiety —(CH₂)_(n)-L-(CH₂)_(m)— is a double bond, the bond

between said ring carbon atom which is bound to the moiety—(CH₂)_(n)-L-(CH₂)_(m)— and the ring atom X₅ is a single bond, and thebond

between the ring atoms X₄ and X₅ is a double bond.

Accordingly, it is particularly preferred that ring B has the followingstructure:

wherein the ring atom X₁ is C(R^(X2)), and the ring atoms X₄ and X₅ areeach C(R^(X3)).

Each R^(X1) is independently selected from hydrogen, C₁₋₅ alkyl,—CO(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-aryl, and —(C₀₋₃ alkylene)-heteroaryl,wherein the aryl comprised in said —(C₀₋₃ alkylene)-aryl and theheteroaryl comprised in said —(C₀₋₃alkylene)-heteroaryl are eachoptionally substituted with one or more (e.g., one, two, or three)groups R^(X11).

Preferably, each R^(X1) is independently selected from hydrogen, C₁₋₅alkyl, —(C₀₋₃ alkylene)-aryl, and —(C₀₋₃ alkylene)-heteroaryl, whereinthe aryl comprised in said —(C₀₋₃ alkylene)-aryl and the heteroarylcomprised in said —(C₀₋₃ alkylene)-heteroaryl are each optionallysubstituted with one or more (e.g., one, two, or three) groups R^(X11).More preferably, each R^(X1) is independently selected from hydrogen,C₁₋₅ alkyl, and —(C₀₋₃ alkylene)-phenyl, wherein the phenyl comprised insaid —(C₀₋₃ alkylene)-phenyl is optionally substituted with one or more(e.g., one, two, or three) groups R. Even more preferably, each R^(X1)is independently selected from hydrogen and C₁₋₅ alkyl. Yet even morepreferably, each R^(X1) is independently selected from hydrogen, methyland ethyl. Still more preferably, each R^(X1) is independently selectedfrom methyl and ethyl. Most preferably, each R^(X1) is methyl.

R^(X2) is selected from hydrogen, C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅haloalkyl), —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃,—(C₀₋₃ alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl).

Preferably, R^(X2) is selected from hydrogen, C₁₋₅ alkyl, C₂₋₅ alkenyl,C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH, —O(C₁₋₅alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅ alkyl),—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), halogen, C₁₋₅ haloalkyl, —O—(C₁₋₅haloalkyl), —CF₃, —CN, —NO₂, —CHO, —CO—(C₁₋₅ alkyl), —COOH, —CO—O—(C₁₋₅alkyl), —O—CO—(C₁₋₅ alkyl), —CO—NH₂, —CO—NH(C₁₋₅ alkyl), —CO—N(C₁₋₅alkyl)(C₁₋₅ alkyl), —NH—CO—(C₁₋₅ alkyl), —N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl),—SO₂—NH₂, —SO₂—NH(C₁₋₅ alkyl), —SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl),—NH—SO₂—(C₁₋₅ alkyl), and —N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl). Morepreferably, R^(X2) is selected from hydrogen, C₁₋₅ alkyl, C₂₋₅ alkenyl,C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH, —O(C₁₋₅alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅ alkyl),—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), halogen, C₁₋₅ haloalkyl, —CF₃, and —CN. Evenmore preferably, R^(X2) is selected from hydrogen, C₁₋₄ alkyl, —OH,—O(C₁₋₄ alkyl), —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)(C₁₋₄ alkyl),halogen, —CF₃, and —CN. Most preferably, R^(X2) is hydrogen.

The two groups R^(X3) (which are present if X⁴ and X⁵ are eachC(R^(X3))) are either mutually linked (i.e., joined) to form, togetherwith the ring carbon atoms that they are attached to (i.e., the ringcarbon atoms in positions X⁴ and X⁵), a 5- or 6-membered cyclyl groupwhich is optionally substituted with one or more (e.g., one, two, orthree) groups R^(X31), or the two groups R^(X3) are each independentlyselected from hydrogen, C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —OH,—O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH, —O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl),—SH, —S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅ alkyl), —N(C₁₋₅ alkyl)(C₁₋₅ alkyl),halogen, C₁₋₅ haloalkyl, —O—(C₁₋₅ haloalkyl), —CF₃, —CN, —NO₂, —CHO,—CO—(C₁₋₅ alkyl), —COOH, —CO—O—(C₁₋₅ alkyl), —O—CO—(C₁₋₅alkyl), —CO—NH₂,—CO—NH(C₁₋₅ alkyl), —CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —NH—CO—(C₁₋₅ alkyl),—N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —SO₂—NH₂, —SO₂—NH(C₁₋₅ alkyl),—SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —NH—SO₂—(C₁₋₅ alkyl), and —N(C₁₋₅alkyl)-SO₂—(C₁₋₅ alkyl). It is preferred that the two groups R^(X3) aremutually linked.

If the two groups R^(X3) are not mutually linked, it is preferred thatthey are each independently selected from hydrogen, C₁₋₅ alkyl, C₂₋₅alkenyl, C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH,—O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅alkyl), —N(C₁₋₅ alkyl)(C₁₋₅ alkyl), halogen, C₁₋₅ haloalkyl, —O—(C₁₋₅haloalkyl), —CF₃, —CN, —NO₂, —CHO, —CO—(C₁₋₅ alkyl), —COOH, —CO—O—(C₁₋₅alkyl), —O—CO—(C₁₋₅ alkyl), —CO—NH₂, —CO—NH(C₁₋₅ alkyl), —CO—N(C₁₋₅alkyl)(C₁₋₅ alkyl), —NH—CO—(C₁₋₅ alkyl), —N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl),—SO₂—NH₂, —SO₂—NH(C₁₋₅ alkyl), —SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl),—NH—SO₂—(C₁₋₅ alkyl), and —N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl), morepreferably from hydrogen, C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —OH,—O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH, alkylene)-O(C₁₋₅ alkyl), —SH,—S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅ alkyl), —N(C₁₋₅ alkyl)(C₁₋₅ alkyl),halogen, C₁₋₅ haloalkyl, —CF₃, and —CN, and even more preferably fromhydrogen, C₁₋₄ alkyl, —OH, —O(C₁₋₄ alkyl), —NH₂, —NH(C₁₋₄ alkyl),—N(C₁₋₄ alkyl)(C₁₋₄ alkyl), halogen, —CF₃, and —CN.

If the two groups R^(X3) are mutually linked to form, together with thering carbon atoms that they are attached to, a 5- or 6-membered cyclylgroup which is optionally substituted with one or more groups R^(X31),it is preferred that said cyclyl group is a 5- or 6-membered cycloalkylgroup (e.g., cyclopentyl or cyclohexyl), a 5- or 6-membered cycloalkenylgroup (e.g., cyclopentenyl, cyclopentadienyl, cyclohexenyl, orcyclohexadienyl), a phenyl group, a 5- or 6-membered heterocycloalkylgroup, a 5- or 6-membered heterocycloalkenyl group, or a 5- or6-membered heteroaryl group, wherein each one of the aforementionedgroups is optionally substituted with one or more (e.g., one, two, orthree) groups R^(X31). More preferably, said cyclyl group is a 5- or6-membered cycloalkyl group (e.g., cyclopentyl or cyclohexyl), a 5- or6-membered cycloalkenyl group (e.g., cyclopentenyl, cyclopentadienyl,cyclohexenyl, or cyclohexadienyl), or a phenyl group, wherein each oneof the aforementioned groups is optionally substituted with one or more(e.g., one, two, or three) groups R^(X31). Even more preferably, saidcyclyl group is a phenyl group, wherein said phenyl group is optionallysubstituted with one or more (e.g., one, two, or three) groups R^(X31).It will be understood that the cyclyl group (including any of theaforementioned preferred cyclyl groups) is formed from the two groupsR^(X3) and the ring carbon atoms (in positions X⁴ and X⁵) that thesegroups R^(X3) are attached to, i.e., the corresponding cyclyl group isfused to the ring containing the ring atoms X¹ to X⁵.

It is particularly preferred that the two groups R^(X3) are mutuallylinked to form, together with the ring carbon atoms that they areattached to, a 5- or 6-membered cycloalkyl group, a 5- or 6-memberedcycloalkenyl group, or a phenyl group, wherein said cycloalkyl group,said cycloalkenyl group, and said phenyl group are each optionallysubstituted with one or more (e.g., one, two, or three) groups R^(X31).It is even more preferred that the ring atoms X⁴ and X⁵ are eachC(R^(X3)) and are connected by a double bond, thus forming a moiety—C(R^(X3))═C(R^(X3))—, and that the two groups R^(X3) are mutuallylinked to form, together with the ring carbon atoms that they areattached to, a cyclopentenyl group, a cyclohexenyl group, or a phenylgroup, wherein said cyclopentenyl group, said cyclohexenyl group, andsaid phenyl group are each optionally substituted with one or more(e.g., one, two, or three) groups R^(X31). It is still more preferredthat the ring atoms X⁴ and X⁵ are each C(R^(X3)) and are connected by adouble bond, thus forming a moiety —C(R^(X3))═C(R^(X3))—, and that thetwo groups R^(X3) are mutually linked to form, together with the ringcarbon atoms that they are attached to, a phenyl group, wherein saidphenyl group is optionally substituted with one or more (e.g., one, two,or three) groups R^(X31).

Each R^(X11) is independently selected from C₁₋₅ alkyl, C₂₋₅ alkenyl,C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl),—(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-OH, alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅haloalkyl), —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃,—(C₀₋₃ alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl).

Preferably, each R^(X11) is independently selected from C₁₋₅ alkyl, C₂₋₅alkenyl, C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH,—O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅alkyl), —N(C₁₋₅ alkyl)(C₁₋₅ alkyl), halogen, C₁₋₅ haloalkyl, haloalkyl),—CF₃, —CN, —NO₂, —CHO, —CO—(C₁₋₅ alkyl), —COOH, —CO—O—(C₁₋₅ alkyl),—O—CO—(C₁₋₅ alkyl), —CO—NH₂, —CO—NH(C₁₋₅ alkyl), —CO—N(C₁₋₅ alkyl)(C₁₋₅alkyl), —NH—CO—(C₁₋₅ alkyl), —N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —SO₂—NH₂,—SO₂—NH(C₁₋₅ alkyl), —SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —NH—SO₂—(C_(1z)alkyl), and —N(C₁₋₅ alkyl)-SO₂—(C_(1z) alkyl). More preferably, eachR^(X11) is independently selected from C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅alkynyl, —OH, —O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH, —O(C₁₋₅alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅ alkyl),—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), halogen, C₁₋₅ haloalkyl, —CF₃, and —CN. Evenmore preferably, each R^(X11) is independently selected from C₁₋₄ alkyl,—OH, —O(C₁₋₄ alkyl), —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)(C₁₋₄ alkyl),halogen, —CF₃, and —CN.

Each R^(X31) is independently selected from C₁₋₅ alkyl, C₂₋₅ alkenyl,C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl),—(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃alkylene)-(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃alkylene)-CF₃, —(C₀₋₃ alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃alkylene)-CHO, —(C₀₋₃ alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH,—(C₀₋₃ alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl),—(C₀₋₃ alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl).

Preferably, each R^(X31) is independently selected from C₁₋₅ alkyl, C₂₋₅alkenyl, C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH,—O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅alkyl), —N(C₁₋₅ alkyl)(C₁₋₅ alkyl), halogen, C₁₋₅ haloalkyl, —O—(C₁₋₅haloalkyl), —CF₃, —CN, —NO₂, —CHO, —CO—(C₁₋₅ alkyl), —COOH, —CO—O—(C₁₋₅alkyl), —O—CO—(C_(1z) alkyl), —CO—NH₂, —CO—NH(C₁₋₅ alkyl), —CO—N(C₁₋₅alkyl)(C₁₋₅ alkyl), —NH—CO—(C₁₋₅ alkyl), —N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl),—SO₂—NH₂, —SO₂—NH(C₁₋₅ alkyl), —SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl),—NH—SO₂—(C₁₋₅ alkyl), and —N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl). Morepreferably, each R^(X31) is independently selected from C₁₋₅ alkyl, C₂₋₅alkenyl, C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH,alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅ alkyl),—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), halogen, C₁₋₅ haloalkyl, —CF₃, and —CN. Evenmore preferably, each R^(X31) is independently selected from C₁₋₄ alkyl,—OH, —O(C₁₋₄ alkyl), —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)(C₁₋₄ alkyl),halogen, —CF₃, and —CN.

Ring B is attached to the remainder of the compound of formula (I), i.e.to the moiety —(CH₂)_(n)-L-(CH₂)_(m)— comprised in the compound offormula (I), via the ring carbon atom that is marked with an asterisk(*) or, if X₄ and X₅ are each C(R⁷³) and the two groups R^(X3) aremutually linked to form, together with the ring carbon atoms that theyare attached to, a 5- or 6-membered cyclyl group (which is optionallysubstituted with one or more groups R^(X31)), then ring B may also beattached to the remainder of the compound of formula (I) via any ringcarbon atom of said 5- or 6-membered cyclyl group.

If X₄ and X₅ are each C(R^(X3)) and the two groups R^(X3) are mutuallylinked to form, together with the ring carbon atoms that they areattached to, a 6-membered cyclyl group (including any one of thespecific or preferred 6-membered groups described herein, such as phenylor cyclohexenyl) which is optionally substituted with one or more groupsR^(X31), then it is preferred that ring B is attached either via thering carbon atom that is marked with an asterisk or via a ring carbonatom in the same position of the 6-membered cyclyl group as in compound30.

It is particularly preferred that ring B is attached via the ring carbonatom that is marked with an asterisk (*). In this case, the compound offormula (I) has the following structure:

If X₄ and X₅ are each C(R^(X3)) and the two groups R^(X3) are mutuallylinked to form, together with the ring carbon atoms that they areattached to, a 6-membered cyclyl group (including any one of thespecific or preferred 6-membered groups described herein above, such asphenyl or cyclohexenyl) which is optionally substituted with one or moregroups R^(X31), then it is also particularly preferred that ring B isattached via a ring carbon atom in the same position of the 6-memberedcyclyl group as in compound 30. Corresponding preferred examples of thecompound of formula (I) are illustrated in the following:

Ring A is aryl or heteroaryl, wherein said aryl and said heteroaryl areeach optionally substituted with one or more (e.g., one, two, or three)groups R^(A).

If ring A is aryl (which is optionally substituted with one or moregroups R^(A)), it is preferred that said aryl is phenyl.

If ring A is heteroaryl (which is optionally substituted with one ormore groups R^(A)), it is preferred that said heteroaryl is selectedfrom the heteroaryl groups specified in the subsequent paragraph, morepreferably from 1,4-benzodioxanyl (particularly 1,4-benzodioxan-6-yl),benzoxanyl (particularly 1-benzoxan-6-yl), 1,3-benzodioxolanyl(particularly 1,3-benzodioxolan-5-yl), benzoxolanyl (particularly1-benzoxolan-5-yl), 1,5-benzodioxepanyl (particularly1,5-benzodioxepan-7-yl), and benzoxepanyl (particularly1-benzoxepan-7-yl), and is even more preferably selected from1,4-benzodioxanyl (particularly 1,4-benzodioxan-6-yl), benzoxanyl(particularly 1-benzoxan-6-yl), 1,3-benzodioxolanyl (particularly1,3-benzodioxolan-5-yl), benzoxolanyl (particularly 1-benzoxolan-5-yl),and 1,5-benzodioxepanyl (particularly 1,5-benzodioxepan-7-yl).

Preferably, ring A is selected from 1,4-benzodioxanyl (particularly1,4-benzodioxan-6-yl), benzoxanyl (particularly 1-benzoxan-6-yl),1,3-benzodioxolanyl (particularly 1,3-benzodioxolan-5-yl), benzoxolanyl(particularly 1-benzoxolan-5-yl), 1,5-benzodioxepanyl (particularly1,5-benzodioxepan-7-yl), benzodioxepanyl (particularly1-benzodioxepan-7-yl), phenyl, and a 5- or 6-membered monocyclicheteroaryl (such as, e.g., pyridinyl (particularly pyridin-3-yl) oroxadiazolyl (particularly 1,2,4-oxadiazolyl or 1,3,4-oxadiazolyl)),wherein each of the aforementioned groups is optionally substituted withone or more (e.g., one, two, or three) groups R^(A). More preferably,ring A is selected from 1,4-benzodioxanyl (particularly1,4-benzodioxan-6-yl), benzoxanyl (particularly 1-benzoxan-6-yl),1,3-benzodioxolanyl (particularly 1,3-benzodioxolan-5-yl), benzoxolanyl(particularly 1-benzoxolan-5-yl), 1,5-benzodioxepanyl (particularly1,5-benzodioxepan-7-yl), and phenyl, wherein each of the aforementionedgroups is optionally substituted with one or more (e.g., one, two, orthree) groups R^(A). Even more preferably, ring A is selected from1,4-benzodioxan-6-yl, 1-benzoxan-6-yl, and 4-(C₁₋₅ alkoxy)-phenyl(particularly 4-methoxyphenyl), wherein the phenyl moiety comprised insaid 1,4-benzodioxan-6-yl or in said 1-benzoxan-6-yl is optionallysubstituted with one or more (e.g., one or two) groups R^(A). Yet evenmore preferably, ring A is selected from 1,4-benzodioxan-6-yl,1-benzoxan-6-yl, and 4-methoxyphenyl.

Each R^(A) is independently selected from C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃alkylene)-(C₁₋₅haloalkyl), —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃,—(C₀₋₃ alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-cycloalkyl, —(C₀₋₃ alkylene)-O-cycloalkyl, —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-cycloalkyl, alkylene)-heterocycloalkyl,—(C₀₋₃ alkylene)-O-heterocycloalkyl, and —(C₀₋₃ alkylene)-O(C₁₋₅alkylene)-heterocycloalkyl.

Preferably, each R^(A) is independently selected from C₁₋₅ alkyl, C₂₋₅alkenyl, C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH,—O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅alkyl), —N(C₁₋₅ alkyl)(C₁₋₅ alkyl), halogen, C₁₋₅ haloalkyl, —O—(C₁₋₅haloalkyl), —CF₃, —CN, —NO₂, —CHO, —CO—(C₁₋₅ alkyl), —COOH, —CO—O—(C₁₋₅alkyl), —O—CO—(C₁₋₅ alkyl), —CO—NH₂, —CO—NH(C₁₋₅ alkyl), —CO—N(C₁₋₅alkyl)(C₁₋₅ alkyl), —NH—CO—(C₁₋₅ alkyl), —N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl),—SO₂—NH₂, —SO₂—NH(C₁₋₅ alkyl), —SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl),—NH—SO₂—(C₁₋₅ alkyl), —N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl), cycloalkyl,—O-cycloalkyl, —O—(C₁₋₅ alkylene)-cycloalkyl, heterocycloalkyl,—O-heterocycloalkyl, and —O—(C₁₋₅ alkylene)-heterocycloalkyl. Morepreferably, each R^(A) is independently selected from C₁₋₅ alkyl, C₂₋₅alkenyl, C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl), —O(C₁₋₅ alkylene)-OH,—O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅ alkyl), —NH₂, —NH(C₁₋₅alkyl), —N(C₁₋₅ alkyl)(C₁₋₅ alkyl), halogen, C₁₋₅ haloalkyl, —CF₃, and—CN. Even more preferably, each R^(A) is independently selected fromC₁₋₄ alkyl, —OH, —O(C₁₋₄ alkyl) (particularly —OCH₃), —NH₂, —NH(C₁₋₄alkyl), —N(C₁₋₄ alkyl)(C₁₋₄ alkyl), halogen, —CF₃, and —CN.

L is selected from —CO—N(R^(L1))—, —N(R^(L1))—CO—, —CO—O—, —O—CO—,—C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—, —C(═S)—N(R^(L1))—,—N(R^(L1))—C(═S)—, —N(R^(L1))—CO—N(R^(L1))—, —O—CO—N(R^(L1))—,—N(R^(L1))—CO—O—, —N(R^(L1))—C(═N—R^(L2))—N(R^(L1))—,—O—C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—O—,—S—C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—S—,—N(R^(L1))—C(═S)—N(R^(L1))—, —O—C(═S)—N(R^(L1))—, —N(R^(L1))—C(═S)—O—,—S—CO—N(R^(L1))—, and —N(R^(L1))—CO—S—.

Preferably, L is selected from —CO—N(R^(L1))—, —N(R^(L1))—CO—, —CO—O—,—O—CO—, —N(R^(L1))—CO—N(R^(L1))—, —O—CO—N(R^(L1))—, and—N(R^(L1))—CO—O—. More preferably, L is selected from —CO—N(R^(L1))—,—N(R^(L1))—CO—, —CO—O—, and —O—CO—. Even more preferably, L is—CO—N(R″)— or —N(R^(L1))—CO—;

accordingly, it is particularly preferred that the moiety—(CH₂)_(n)-L-(CH₂)_(m)— comprised in the compound of formula (I) isselected from —(CH₂)_(n)—CO—N(R^(L1))—(CH₂)_(m)— and—(CH₂)_(n)—N(R^(L1))—CO—(CH₂)_(m)—. Most preferably, L is—N(R^(L1))—CO—, wherein said —N(R^(L1))—CO— is bound via its —N(R^(L1))—group to the moiety —(CH₂)_(n)— comprised in the compound of formula(I), and wherein said —N(R^(L1))—CO— is bound via its —CO— group to themoiety —(CH₂)_(n)— comprised in the compound of formula (I);accordingly, it is most preferred that the moiety—(CH₂)_(n)-L-(CH₂)_(m)-comprised in the compound of formula (I) is—(CH₂), —N(R^(L1))—CO—(CH₂)_(n)—.

Each R^(L1) is independently selected from hydrogen and C₁₋₅ alkyl.Preferably, each R^(L1) is independently selected from hydrogen, methyl,and ethyl. More preferably, each R^(L1) is hydrogen.

Each R^(L2) is independently selected from hydrogen, C₁₋₅ alkyl, —CN,and —NO₂. Preferably, each R^(L2) is independently selected fromhydrogen, methyl, ethyl, —CN, and —NO₂. More preferably, each R^(L2) isindependently selected from hydrogen, methyl, ethyl, and —CN. Even morepreferably, each R^(L2) is independently selected from hydrogen, methyl,and ethyl.

n is 0 or 1. Preferably, n is O.

m is 0 or 1, Preferably, m is O.

It is to be understood that n indicates the number of methylene groups—(CH₂)_(n)— that are present between the group L and the ring containingthe ring atoms X₁ to X₅. If n is 0, then group L is directly bound(i.e., bound via a covalent single bond) to the ring containing X₁ toX₅. Likewise, m indicates the number of methylene groups —(CH₂)_(m)—that are present between the group L and the ring group A. If m is 0,then group L is directly bound (i.e., bound via a covalent single bond)to the ring group A.

In accordance with the preferred meanings of L, R^(L1), R^(L2), m and ndescribed above, it is particularly preferred that L is —CO—N(R^(L1))—or —N(R^(L1))—CO—, wherein R^(L1) is selected from hydrogen, methyl, andethyl, and that n and m are each 0. Still more preferably, L is—N(R^(L1))—CO—, wherein R^(L1) is selected from hydrogen, methyl, andethyl, and n and m are each 0. Most preferably, L is —NH—CO—, n is 0,and m is 0. Accordingly, it is most preferred that the moiety—(CH₂)_(n)-L-(CH₂)_(m)— comprised in the compound of formula (I) is—(CH₂)_(n)—NH—CO—(CH₂)_(m)—, wherein n and m are each 0.

The compound of formula (I) may be, for example, any one of thefollowing compounds or a pharmaceutically acceptable salt, solvate orprodrug thereof:

The above-depicted compounds 4-26, 4-1, 29, 30, CeMMEC1, 38, 33, 37, A1,27 and 6 as well as pharmaceutically acceptable salts, solvates andprodrugs thereof are particularly preferred examples of the compound offormula (I). The compounds 4-26, 4-1, 29 and 30 (particularly compound4-26), and pharmaceutically acceptable salts, solvates and prodrugsthereof, are even more preferred.

It will be understood that in the above-depicted compounds 36, 4-1, 4-2,4-3, 4-4, 4-10, 4-13, 4-14, 4-16, 4-17, 4-24, 4-25, 4-26, 4-28, 4-31,4-32 and 4-33, the nitrogen atom in the linker group L (which isdepicted as —N—), is substituted by a hydrogen atom (i.e., is present as—NH—).

In one embodiment of the compound of formula (I), X₂ is C(═O) and X₃ isN(R^(X1)), the moiety —(CH₂)_(n)-L-(CH₂)_(m)— is—(CH₂)—CO—N(R^(L1))—(CH₂)_(m)—, and the further groups/variablescomprised in formula (I) have the same meanings, including the samepreferred meanings, as described and defined herein above.

In a further embodiment of the compound of formula (I), X₂ is C(═O) andX₃ is N(R^(X1)), the moiety-(CH₂)_(n)-L-(CF¹ ₂)_(m)— is—(CH₂)_(n)—N(R^(L1))—CO—(CH₂)_(m)—, and the further groups/variablescomprised in formula (I) have the same meanings, including the samepreferred meanings, as described and defined herein above.

In a further embodiment of the compound of formula (I), X₂ is N(R^(X1))and X₃ is C(═O), the moiety —(CH₂)_(n)—I—(CH₂)_(m)— is—(CH₂)_(n)—CO—N(R^(L1))—(CH₂)_(m)—, and the further groups/variablescomprised in formula (I) have the same meanings, including the samepreferred meanings, as described and defined herein above.

In a further embodiment of the compound of formula (I), X₂ is N(R^(X1))and X₃ is C(═O), the moiety —(CH₂)_(n)-L-(CH₂)_(m)— is—(CH₂)_(n)—N(R^(L1))—CO—(CH₂)_(m)—, and the further groups/variablescomprised in formula (I) have the same meanings, including the samepreferred meanings, as described and defined herein above.

The compounds of formula (I) can be prepared by methods known in thefield of synthetic chemistry. For example, these compounds can beprepared in accordance with or in analogy to the synthetic routedescribed in Example 1.

The following definitions apply throughout the present specification,unless specifically indicated otherwise.

The term “hydrocarbon group” refers to a group consisting of carbonatoms and hydrogen atoms.

The term “alicyclic” is used in connection with cyclic groups anddenotes that the corresponding cyclic group is non-aromatic.

As used herein, the term “alkyl” refers to a monovalent saturatedacyclic (i.e., non-cyclic) hydrocarbon group which may be linear orbranched. Accordingly, an “alkyl” group does not comprise anycarbon-to-carbon double bond or any carbon-to-carbon triple bond. A“C₁₋₅ alkyl” denotes an alkyl group having 1 to 5 carbon atoms.Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g.,n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, ortert-butyl). Unless defined otherwise, the term “alkyl” preferablyrefers to C₁₋₄ alkyl, more preferably to methyl or ethyl, and even morepreferably to methyl.

As used herein, the term “alkenyl” refers to a monovalent unsaturatedacyclic hydrocarbon group which may be linear or branched and comprisesone or more (e.g., one or two) carbon-to-carbon double bonds while itdoes not comprise any carbon-to-carbon triple bond. The term “C₂₋₅alkenyl” denotes an alkenyl group having 2 to 5 carbon atoms. Preferredexemplary alkenyl groups are ethenyl, propenyl (e.g., prop-1-en-1-yl,prop-1-en-2-yl, or prop-2-en-1-yl), butenyl, butadienyl (e.g.,buta-1,3-dien-1-yl or buta-1,3-dien-2-yl), pentenyl, or pentadienyl(e.g., isoprenyl). Unless defined otherwise, the term “alkenyl”preferably refers to C₂₋₄ alkenyl.

As used herein, the term “alkynyl” refers to a monovalent unsaturatedacyclic hydrocarbon group which may be linear or branched and comprisesone or more (e.g., one or two) carbon-to-carbon triple bonds andoptionally one or more carbon-to-carbon double bonds. The term “C₂₋₅alkynyl” denotes an alkynyl group having 2 to 5 carbon atoms. Preferredexemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), orbutynyl. Unless defined otherwise, the term “alkynyl” preferably refersto C₂₋₄ alkynyl.

As used herein, the term “alkylene” refers to an alkanediyl group, i.e.a divalent saturated acyclic hydrocarbon group which may be linear orbranched. A “C₁₋₅ alkylene” denotes an alkylene group having 1 to 5carbon atoms, and the term “C₀₋₃ alkylene” indicates that a covalentbond (corresponding to the option “C₀ alkylene”) or a C₁₋₃ alkylene ispresent. Preferred exemplary alkylene groups are methylene (—CH₂—),ethylene (e.g., —CH₂—CH₂— or —CH(—CH₃)—), propylene (e.g.,—CH₂—CH₂—CH₂—, —CH(—CH₂—CH₃)—, —CH₂—CH(—CH₃)—, or —CH(—CH₃)—CH₂—), orbutylene (e.g., —CH₂—CH₂—CH₂—CH₂—). Unless defined otherwise, the term“alkylene” preferably refers to C₁₋₄ alkylene (including, in particular,linear C₁₋₄ alkylene), more preferably to methylene or ethylene, andeven more preferably to methylene.

As used herein, the term “alkoxy” refers to an —O-alkyl group, whereinthe alkyl moiety comprised in this group is as defined above.

As used herein, the term “carbocyclyl” refers to a hydrocarbon ringgroup, including monocyclic rings as well as bridged ring, spiro ringand/or fused ring systems (which may be composed, e.g., of two or threerings), wherein said ring group may be saturated, partially unsaturated(i.e., unsaturated but not aromatic) or aromatic. Unless definedotherwise, “carbocyclyl” preferably refers to aryl, cycloalkyl orcycloalkenyl.

As used herein, the term “heterocyclyl” refers to a ring group,including monocyclic rings as well as bridged ring, Spiro ring and/orfused ring systems (which may be composed, e.g., of two or three rings),wherein said ring group comprises one or more (such as, e.g., one, two,three, or four) ring heteroatoms independently selected from O, S and N,and the remaining ring atoms are carbon atoms, wherein one or more Sring atoms (if present) and/or one or more N ring atoms (if present) mayoptionally be oxidized, wherein one or more carbon ring atoms mayoptionally be oxidized (i.e., to form an oxo group), and further whereinsaid ring group may be saturated, partially unsaturated (i.e.,unsaturated but not aromatic) or aromatic.

For example, each heteroatom-containing ring comprised in said ringgroup may contain one or two O atoms and/or one or two S atoms (whichmay optionally be oxidized) and/or one, two, three or four N atoms(which may optionally be oxidized), provided that the total number ofheteroatoms in the corresponding heteroatom-containing ring is 1 to 4and that there is at least one carbon ring atom (which may optionally beoxidized) in the corresponding heteroatom-containing ring. Unlessdefined otherwise, “heterocyclyl” preferably refers to heteroaryl,heterocycloalkyl or heterocycloalkenyl.

As used herein, the term “cyclyl” refers to a carbocyclyl or aheterocyclyl, as defined herein above.

As used herein, the term “aryl” refers to an aromatic hydrocarbon ringgroup, including monocyclic aromatic rings as well as bridged ringand/or fused ring systems containing at least one aromatic ring (e.g.,ring systems composed of two or three fused rings, wherein at least oneof these fused rings is aromatic; or bridged ring systems composed oftwo or three rings, wherein at least one of these bridged rings isaromatic). “Aryl” may, e.g., refer to phenyl, naphthyl, dialinyl (i.e.,1,2-dihydronaphthyl), tetralinyl (i.e., 1,2,3,4-tetrahydronaphthyl),indanyl, indenyl (e.g., 1H-indenyl), anthracenyl, phenanthrenyl,9H-fluorenyl, or azulenyl. Unless defined otherwise, an “aryl”preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms,even more preferably refers to phenyl or naphthyl, and most preferablyrefers to phenyl.

As used herein, the term “heteroaryl” refers to an aromatic ring group,including monocyclic aromatic rings as well as bridged ring and/or fusedring systems containing at least one aromatic ring (e.g., ring systemscomposed of two or three fused rings, wherein at least one of thesefused rings is aromatic; or bridged ring systems composed of two orthree rings, wherein at least one of these bridged rings is aromatic),wherein said aromatic ring group comprises one or more (such as, e.g.,one, two, three, or four) ring heteroatoms independently selected fromO, S and N, and the remaining ring atoms are carbon atoms, wherein oneor more S ring atoms (if present) and/or one or more N ring atoms (ifpresent) may optionally be oxidized, and further wherein one or morecarbon ring atoms may optionally be oxidized (i.e., to form an oxogroup). For example, each heteroatom-containing ring comprised in saidaromatic ring group may contain one or two O atoms and/or one or two Satoms (which may optionally be oxidized) and/or one, two, three or fourN atoms (which may optionally be oxidized), provided that the totalnumber of heteroatoms in the corresponding heteroatom-containing ring is1 to 4 and that there is at least one carbon ring atom (which mayoptionally be oxidized) in the corresponding heteroatom-containing ring.“Heteroaryl” may, e.g., refer to thienyl (i.e., thiophenyl),benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e.,furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g.,2H-1-benzopyranyl or 4H-1-benzopyranyl), isochromenyl (e.g.,1H-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl(e.g., 1H-pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl;e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyrazinyl, pyrimidinyl,pyridazinyl, indolyl (e.g., 3H-indolyl), isoindolyl, indazolyl,indolizinyl, purinyl, quinolyl, isoquinolyl, phthalazinyl,naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl(e.g., [1,10]phenanthrolinyl, [1,7]phenanthrolinyl, or[4,7]phenanthrolinyl), phenazinyl, thiazolyl, isothiazolyl,phenothiazinyl, oxazolyl, isoxazolyl, oxadiazolyl (e.g.,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl (i.e., furazanyl), or1,3,4-oxadiazolyl), thiadiazolyl (e.g., 1,2,4-thiadiazolyl,1,2,5-thiadiazolyl, or 1,3,4-thiadiazolyl), phenoxazinyl,pyrazolo[1,5-a]pyrimidinyl (e.g., pyrazolo[1,5-a]pyrimidin-3-yl),1,2-benzoisoxazol-3-yl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl,benzisoxazolyl, benzimidazolyl, benzo[b]thiophenyl (i.e., benzothienyl),triazolyl (e.g., 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl,1H-1,2,4-triazolyl, or 4H-1,2,4-triazolyl), benzotriazolyl,1H-tetrazolyl, 2H-tetrazolyl, triazinyl (e.g., 1,2,3-triazinyl,1,2,4-triazinyl, or 1,3,5-triazinyl), furo[2,3-c]pyridinyl,dihydrofuropyridinyl (e.g., 2,3-dihydrofuro[2,3-c]pyridinyl or1,3-dihydrofuro[3,4-c]pyridinyl), imidazopyridinyl (e.g.,imidazo[1,2-a]pyridinyl or imidazo[3,2-a]pyridinyl), quinazolinyl,thienopyridinyl, tetrahydrothienopyridinyl (e.g.,4,5,6,7-tetrahydrothieno[3,2-c]pyridinyl), dibenzofuranyl,1,3-benzodioxolyl, benzodioxanyl (e.g., 1,3-benzodioxanyl or1,4-benzodioxanyl), or coumarinyl. Unless defined otherwise, the term“heteroaryl” preferably refers to a 5 to 14 membered (more preferably 5to 10 membered) monocyclic ring or fused ring system comprising one ormore (e.g., one, two, three or four) ring heteroatoms independentlyselected from O, S and N, wherein one or more S ring atoms (if present)and/or one or more N ring atoms (if present) are optionally oxidized,and wherein one or more carbon ring atoms are optionally oxidized; evenmore preferably, a “heteroaryl” refers to a 5 or 6 membered monocyclicring comprising one or more (e.g., one, two or three) ring heteroatomsindependently selected from O, S and N, wherein one or more S ring atoms(if present) and/or one or more N ring atoms (if present) are optionallyoxidized, and wherein one or more carbon ring atoms are optionallyoxidized. Moreover, unless defined otherwise, the term “heteroaryl”particularly preferably refers to pyridinyl (e.g., 2-pyridyl, 3-pyridyl,or 4-pyridyl), imidazolyl, thiazolyl, 1H-tetrazolyl, 2H-tetrazolyl,thienyl (i.e., thiophenyl), or pyrimidinyl.

As used herein, the term “cycloalkyl” refers to a saturated hydrocarbonring group, including monocyclic rings as well as bridged ring, Spiroring and/or fused ring systems (which may be composed, e.g., of two orthree rings; such as, e.g., a fused ring system composed of two or threefused rings). “Cycloalkyl” may, e.g., refer to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e.,decahydronaphthyl), or adamantyl. Unless defined otherwise, “cycloalkyl”preferably refers to a C₃₋₁₁ cycloalkyl, and more preferably refers to aC₃₋₇ cycloalkyl. A particularly preferred “cycloalkyl” is a monocyclicsaturated hydrocarbon ring having 3 to 7 ring members. Moreover, unlessdefined otherwise, the term “cycloalkyl” even more preferably refers tocyclohexyl or cyclopropyl, and yet even more preferably refers tocyclohexyl.

As used herein, the term “heterocycloalkyl” refers to a saturated ringgroup, including monocyclic rings as well as bridged ring, Spiro ringand/or fused ring systems (which may be composed, e.g., of two or threerings; such as, e.g., a fused ring system composed of two or three fusedrings), wherein said ring group contains one or more (such as, e.g.,one, two, three, or four) ring heteroatoms independently selected fromO, S and N, and the remaining ring atoms are carbon atoms, wherein oneor more S ring atoms (if present) and/or one or more N ring atoms (ifpresent) may optionally be oxidized, and further wherein one or morecarbon ring atoms may optionally be oxidized (i.e., to form an oxogroup). For example, each heteroatom-containing ring comprised in saidsaturated ring group may contain one or two O atoms and/or one or two Satoms (which may optionally be oxidized) and/or one, two, three or fourN atoms (which may optionally be oxidized), provided that the totalnumber of heteroatoms in the corresponding heteroatom-containing ring is1 to 4 and that there is at least one carbon ring atom (which mayoptionally be oxidized) in the corresponding heteroatom-containing ring.“Heterocycloalkyl” may, e.g., refer to aziridinyl, azetidinyl,pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl,azepanyl, diazepanyl (e.g., 1,4-diazepanyl), oxazolidinyl,isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl (e.g.,morpholin-4-yl), thiomorpholinyl (e.g., thiomorpholin-4-yl), oxazepanyl,oxiranyl, oxetanyl, tetrahydrofuranyl, 1,3-dioxolanyl,tetrahydropyranyl, 1,4-dioxanyl, oxepanyl, thiiranyl, thietanyl,tetrahydrothiophenyl (i.e., thiolanyl), 1,3-dithiolanyl, thianyl,thiepanyl, decahydroquinolinyl, decahydroisoquinolinyl, or2-oxa-5-aza-bicyclo[2.2.1]hept-5-yl. Unless defined otherwise,“heterocycloalkyl” preferably refers to a 3 to 11 membered saturatedring group, which is a monocyclic ring or a fused ring system (e.g., afused ring system composed of two fused rings), wherein said ring groupcontains one or more (e.g., one, two, three, or four) ring heteroatomsindependently selected from O, S and N, wherein one or more S ring atoms(if present) and/or one or more N ring atoms (if present) are optionallyoxidized, and wherein one or more carbon ring atoms are optionallyoxidized; more preferably, “heterocycloalkyl” refers to a 5 to 7membered saturated monocyclic ring group containing one or more (e.g.,one, two, or three) ring heteroatoms independently selected from O, Sand N, wherein one or more S ring atoms (if present) and/or one or moreN ring atoms (if present) are optionally oxidized, and wherein one ormore carbon ring atoms are optionally oxidized. Moreover, unless definedotherwise, “heterocycloalkyl” even more preferably refers totetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl,or tetrahydrofuranyl.

As used herein, the term “cycloalkenyl” refers to an unsaturatedalicyclic (non-aromatic) hydrocarbon ring group, including monocyclicrings as well as bridged ring, spiro ring and/or fused ring systems(which may be composed, e.g., of two or three rings; such as, e.g., afused ring system composed of two or three fused rings), wherein saidhydrocarbon ring group comprises one or more (e.g., one or two)carbon-to-carbon double bonds and does not comprise any carbon-to-carbontriple bond. “Cycloalkenyl” may, e.g., refer to cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl,cycloheptenyl, or cycloheptadienyl. Unless defined otherwise,“cycloalkenyl” preferably refers to a C₃₋₁₁ cycloalkenyl, and morepreferably refers to a C₃₋₇ cycloalkenyl. A particularly preferred“cycloalkenyl” is a monocyclic unsaturated alicyclic hydrocarbon ringhaving 3 to 7 ring members and containing one or more (e.g., one or two;preferably one) carbon-to-carbon double bonds.

As used herein, the term “heterocycloalkenyl” refers to an unsaturatedalicyclic (non-aromatic) ring group, including monocyclic rings as wellas bridged ring, Spiro ring and/or fused ring systems (which may becomposed, e.g., of two or three rings; such as, e.g., a fused ringsystem composed of two or three fused rings), wherein said ring groupcontains one or more (such as, e.g., one, two, three, or four) ringheteroatoms independently selected from 0, S and N, and the remainingring atoms are carbon atoms, wherein one or more S ring atoms (ifpresent) and/or one or more N ring atoms (if present) may optionally beoxidized, wherein one or more carbon ring atoms may optionally beoxidized (i.e., to form an oxo group), and further wherein said ringgroup comprises at least one double bond between adjacent ring atoms anddoes not comprise any triple bond between adjacent ring atoms. Forexample, each heteroatom-containing ring comprised in said unsaturatedalicyclic ring group may contain one or two O atoms and/or one or two Satoms (which may optionally be oxidized) and/or one, two, three or fourN atoms (which may optionally be oxidized), provided that the totalnumber of heteroatoms in the corresponding heteroatom-containing ring is1 to 4 and that there is at least one carbon ring atom (which mayoptionally be oxidized) in the corresponding heteroatom-containing ring.“Heterocycloalkenyl” may, e.g., refer to imidazolinyl (e.g.,2-imidazolinyl (i.e., 4,5-dihydro-1H-imidazolyl), 3-imidazolinyl, or4-imidazolinyl), tetrahydropyridinyl (e.g.,1,2,3,6-tetrahydropyridinyl), dihydropyridinyl (e.g.,1,2-dihydropyridinyl or 2,3-dihydropyridinyl), pyranyl (e.g., 2H-pyranylor 4H-pyranyl), thiopyranyl (e.g., 2H-thiopyranyl or 4H-thiopyranyl),dihydropyranyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrazinyl,dihydroisoindolyl, octahydroquinolinyl (e.g.,1,2,3,4,4a,5,6,7-octahydroquinolinyl), or octahydroisoquinolinyl (e.g.,1,2,3,4,5,6,7,8-octahydroisoquinolinyl). Unless defined otherwise,“heterocycloalkenyl” preferably refers to a 3 to 11 membered unsaturatedalicyclic ring group, which is a monocyclic ring or a fused ring system(e.g., a fused ring system composed of two fused rings), wherein saidring group contains one or more (e.g., one, two, three, or four) ringheteroatoms independently selected from O, S and N, wherein one or moreS ring atoms (if present) and/or one or more N ring atoms (if present)are optionally oxidized, wherein one or more carbon ring atoms areoptionally oxidized, and wherein said ring group comprises at least onedouble bond between adjacent ring atoms and does not comprise any triplebond between adjacent ring atoms; more preferably, “heterocycloalkenyl”refers to a 5 to 7 membered monocyclic unsaturated non-aromatic ringgroup containing one or more (e.g., one, two, or three) ring heteroatomsindependently selected from O, S and N, wherein one or more S ring atoms(if present) and/or one or more N ring atoms (if present) are optionallyoxidized, wherein one or more carbon ring atoms are optionally oxidized,and wherein said ring group comprises at least one double bond betweenadjacent ring atoms and does not comprise any triple bond betweenadjacent ring atoms.

As used herein, the term “halogen” refers to fluoro (—F), chloro (—Cl),bromo (—Br), or iodo (—I).

As used herein, the term “haloalkyl” refers to an alkyl groupsubstituted with one or more (preferably 1 to 6, more preferably 1 to 3)halogen atoms which are selected independently from fluoro, chloro,bromo and iodo, and are preferably all fluoro atoms. It will beunderstood that the maximum number of halogen atoms is limited by thenumber of available attachment sites and, thus, depends on the number ofcarbon atoms comprised in the alkyl moiety of the haloalkyl group.“Haloalkyl” may, e.g., refer to —CF₃, —CHF₂, —CH₂F, —CF₂—CH₃, —CH₂—CF₃,—CH₂—CHF₂, —CH₂—CF₂—CH₃, —CH₂—CF₂—CF₃, or —CH(CF₃)₂. A particularlypreferred “haloalkyl” group is —CF₃.

As used herein, the terms “optional”, “optionally” and “may” denote thatthe indicated feature may be present but can also be absent. Wheneverthe term “optional”, “optionally” or “may” is used, the presentinvention specifically relates to both possibilities, i.e., that thecorresponding feature is present or, alternatively, that thecorresponding feature is absent. For example, the expression “X isoptionally substituted with Y” (or “X may be substituted with Y”) meansthat X is either substituted with Y or is unsubstituted. Likewise, if acomponent of a composition is indicated to be “optional”, the inventionspecifically relates to both possibilities, i.e., that the correspondingcomponent is present (contained in the composition) or that thecorresponding component is absent from the composition.

Various groups are referred to as being “optionally substituted” in thisspecification. Generally, these groups may carry one or moresubstituents, such as, e.g., one, two, three or four substituents. Itwill be understood that the maximum number of substituents is limited bythe number of attachment sites available on the substituted moiety.Unless defined otherwise, the “optionally substituted” groups referredto in this specification carry preferably not more than two substituentsand may, in particular, carry only one substituent. Moreover, unlessdefined otherwise, it is preferred that the optional substituents areabsent, i.e. that the corresponding groups are unsubstituted.

A skilled person will appreciate that the substituent groups comprisedin the compounds of formula (I) may be attached to the remainder of therespective compound via a number of different positions of thecorresponding specific substituent group. Unless defined otherwise, thepreferred attachment positions for the various specific substituentgroups are as illustrated in the examples.

As used herein, unless explicitly indicated otherwise or contradicted bycontext, the terms “a”, “an” and “the” are used interchangeably with“one or more” and “at least one”. Thus, for example, a compositioncomprising “a” compound of formula (I) can be interpreted as referringto a composition comprising “one or more” compounds of formula (I).

As used herein, the term “comprising” (or “comprise”, “comprises”,“contain”, “contains”, or “containing”), unless explicitly indicatedotherwise or contradicted by context, has the meaning of “containing,inter alia”, i.e., “containing, among further optional elements, . . .”. In addition thereto, this term also includes the narrower meanings of“consisting essentially of” and “consisting of”. For example, the term“A comprising B and C” has the meaning of “A containing, inter alia, Band C”, wherein A may contain further optional elements (e.g., “Acontaining B, C and D” would also be encompassed), but this term alsoincludes the meaning of “A consisting essentially of B and C” and themeaning of “A consisting of B and C” (i.e., no other components than Band C are comprised in A).

Moreover, unless indicated otherwise, any reference to an industrystandard, a pharmacopeia, or a manufacturer's manual refers to thecorresponding latest version that was available at the priority date(i.e., at the earliest filing date) of the present specification.

The scope of the invention embraces all pharmaceutically acceptable saltforms of the compounds provided herein, particularly the compounds offormula (I), which may be formed, e.g., by protonation of an atomcarrying an electron lone pair which is susceptible to protonation, suchas an amino group, with an inorganic or organic acid, or as a salt of anacid group (such as a carboxylic acid group) with a physiologicallyacceptable cation. Exemplary base addition salts comprise, for example:alkali metal salts such as sodium or potassium salts; alkaline earthmetal salts such as calcium or magnesium salts; zinc salts; ammoniumsalts; aliphatic amine salts such as trimethylamine, triethylamine,dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine,procaine salts, meglumine salts, ethylenediamine salts, or cholinesalts; aralkyl amine salts such as N,N-dibenzylethylenediamine salts,benzathine salts, benethamine salts; heterocyclic aromatic amine saltssuch as pyridine salts, picoline salts, quinoline salts or isoquinolinesalts; quaternary ammonium salts such as tetramethylammonium salts,tetraethylammonium salts, benzyltrimethylammonium salts,benzyltriethylammonium salts, benzyltributylammonium salts,methyltrioctylammonium salts or tetrabutylammonium salts; and basicamino acid salts such as arginine salts, lysine salts, or histidinesalts. Exemplary acid addition salts comprise, for example: mineral acidsalts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts(such as, e.g., sulfate or hydrogensulfate salts), nitrate salts,phosphate salts (such as, e.g., phosphate, hydrogenphosphate, ordihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts,perchlorate salts, borate salts, or thiocyanate salts; organic acidsalts such as acetate, propionate, butyrate, pentanoate, hexanoate,heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate,oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate,citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate,salicylate, ascorbate, pamoate (embonate), camphorate, glucoheptanoate,or pivalate salts; sulfonate salts such as methanesulfonate (mesylate),ethanesulfonate (esylate), 2-hydroxyethanesulfonate (isethionate),benzenesulfonate (besylate), p-toluenesulfonate (tosylate),2-naphthalenesulfonate (napsylate), 3-phenylsulfonate, orcamphorsulfonate salts; glycerophosphate salts; and acidic amino acidsalts such as aspartate or glutamate salts.

Moreover, the scope of the invention embraces the compounds providedherein, particularly the compounds of formula (I), in any solvated form,including, e.g., solvates with water (i.e., as a hydrate) or solvateswith organic solvents such as, e.g., methanol, ethanol or acetonitrile(i.e., as a methanolate, ethanolate or acetonitrilate), or in anycrystalline form (i.e., as any polymorph), or in amorphous form. It isto be understood that such solvates of the compounds provided herein,particularly the compounds of formula (I), also include solvates ofpharmaceutically acceptable salts of the corresponding compounds.

Furthermore, the compounds provided herein, particularly the compoundsof formula (I), may exist in the form of different isomers, inparticular stereoisomers (including, e.g., geometric isomers (orcis/trans isomers), enantiomers and diastereomers) or tautomers. Allsuch isomers of the compounds provided herein are contemplated as beingpart of the present invention, either in admixture or in pure orsubstantially pure form. As for stereoisomers, the invention embracesthe isolated optical isomers of the compounds according to the inventionas well as any mixtures thereof (including, in particular, racemicmixtures/racemates). The racemates can be resolved by physical methods,such as, e.g., fractional crystallization, separation or crystallizationof diastereomeric derivatives, or separation by chiral columnchromatography. The individual optical isomers can also be obtained fromthe racemates via salt formation with an optically active acid followedby crystallization. The present invention further encompasses anytautomers of the compounds provided herein.

The scope of the invention also embraces the compounds provided herein,particularly the compounds of formula (I), in which one or more atomsare replaced by a specific isotope of the corresponding atom. Forexample, the invention encompasses compounds of formula (I), in whichone or more hydrogen atoms (or, e.g., all hydrogen atoms) are replacedby deuterium atoms (i.e., ²H; also referred to as “D”). Accordingly, theinvention also embraces compounds of formula (I) which are enriched indeuterium. Naturally occurring hydrogen is an isotopic mixturecomprising about 99.98 mol-% hydrogen-1 (¹H) and about 0.0156 mol-%deuterium (²H or D). The content of deuterium in one or more hydrogenpositions in the compounds of formula (I) can be increased usingdeuteration techniques known in the art. For example, a compound offormula (I) or a reactant or precursor to be used in the synthesis ofthe compound of formula (I) can be subjected to an H/D exchange reactionusing, e.g., heavy water (D₂O). Further suitable deuteration techniquesare described in: Atzrodt J et al., Bioorg Med Chem, 20(18), 5658-5667,2012; William J S et al., Journal of Labelled Compounds andRadiopharmaceuticals, 53(11-12), 635-644, 2010; or Modvig A et al., JOrg Chem, 79, 5861-5868, 2014. The content of deuterium can bedetermined, e.g., using mass spectrometry or NMR spectroscopy. Unlessspecifically indicated otherwise, it is preferred that the compound offormula (I) is not enriched in deuterium. Accordingly, the presence ofnaturally occurring hydrogen atoms or ¹H hydrogen atoms in the compoundsof formula (I) is preferred.

The present invention also embraces the compounds provided herein,particularly the compounds of formula (I), in which one or more atomsare replaced by a positron-emitting isotope of the corresponding atom,such as, e.g., ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, ⁷⁷Br, ¹²⁰I and/or ¹²⁴I. Suchcompounds can be used as tracers or imaging probes in positron emissiontomography (PET). The invention thus includes (i) compounds of formula(I), in which one or more fluorine atoms (or, e.g., all fluorine atoms)are replaced by ¹⁸F atoms, (ii) compounds of formula (I), in which oneor more carbon atoms (or, e.g., all carbon atoms) are replaced by ¹¹Catoms, (iii) compounds of formula (I), in which one or more nitrogenatoms (or, e.g., all nitrogen atoms) are replaced by ¹³N atoms, (iv)compounds of formula (I), in which one or more oxygen atoms (or, e.g.,all oxygen atoms) are replaced by ¹⁵O atoms, (v) compounds of formula(I), in which one or more bromine atoms (or, e.g., all bromine atoms)are replaced by ⁷⁶Br atoms, (vi) compounds of formula (I), in which oneor more bromine atoms (or, e.g., all bromine atoms) are replaced by ⁷⁷Bratoms, (vii) compounds of formula (I), in which one or more iodine atoms(or, e.g., all iodine atoms) are replaced by ¹²⁰I atoms, and (viii)compounds of formula (I), in which one or more iodine atoms (or, e.g.,all iodine atoms) are replaced by ¹²⁴I atoms. In general, it ispreferred that none of the atoms in the compounds of formula (I) arereplaced by specific isotopes.

Pharmaceutically acceptable prodrugs of the compounds provided herein,particularly the compounds of formula (I), are derivatives which havechemically or metabolically cleavable groups and become, by solvolysisor under physiological conditions, the compounds of the invention whichare pharmaceutically active in vivo. Prodrugs of the compounds accordingto the the present invention may be formed in a conventional manner witha functional group of the compounds such as, e.g., with an amino,hydroxy or carboxy group. The prodrug form often offers advantages interms of solubility, tissue compatibility or delayed release in amammalian organism (see, Bundgaard, H., Design of Prodrugs, pp. 7-9,21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives,such as, e.g., esters prepared by reaction of the parent acidic compoundwith a suitable alcohol, or amides prepared by reaction of the parentacid compound with a suitable amine. If a compound of the presentinvention has a carboxyl group, an ester derivative prepared by reactingthe carboxyl group with a suitable alcohol or an amide derivativeprepared by reacting the carboxyl group with a suitable amine isexemplified as a prodrug. An especially preferred ester derivative as aprodrug is methylester, ethylester, n-propylester, isopropylester,n-butylester, isobutylester, tert-butylester, morpholinoethylester,N,N-diethylglycolamidoester or α-acetoxyethylester. If a compound of thepresent invention has a hydroxy group, an acyloxy derivative prepared byreacting the hydroxyl group with a suitable acylhalide or a suitableacid anhydride is exemplified as a prodrug. An especially preferredacyloxy derivative as a prodrug is —OC(═O)—CH₃, —OC(═O)—C₂H₅,—OC(═O)-(tert-Bu), —OC(═O)—O₁₅H₃₁, —OC(═O)-(m-COONa-Ph),—OC(═O)—CH₂CH₂COONa, —O(C═O)—CH(NH₂)CH₃ or —OC(═O)—CH₂—N(CH₃)₂. If acompound of the present invention has an amino group, an amidederivative prepared by reacting the amino group with a suitable acidhalide or a suitable mixed anhydride is exemplified as a prodrug. Anespecially preferred amide derivative as a prodrug is—NHC(═O)—(CH₂)₂OCH₃ or —NHC(═O)—CH(NH₂)CH₃.

The compounds provided herein, including in particular the compounds offormula (I), may be administered as compounds per se or may beformulated as medicaments. The medicaments/pharmaceutical compositionsmay optionally comprise one or more pharmaceutically acceptableexcipients, such as carriers, diluents, fillers, disinteg rants,lubricating agents, binders, colorants, pigments, stabilizers,preservatives, antioxidants, and/or solubility enhancers.

The pharmaceutical compositions may comprise one or more solubilityenhancers, such as, e.g., poly(ethylene glycol), including poly(ethyleneglycol) having a molecular weight in the range of about 200 to about5,000 Da (e.g., PEG 200, PEG 300, PEG 400, or PEG 600), ethylene glycol,propylene glycol, glycerol, a non-ionic surfactant, tyloxapol,polysorbate 80, macrogol-15-hydroxystearate (e.g., Kolliphor® HS 15, CAS70142-34-6), a phospholipid, lecithin, dimyristoyl phosphatidylcholine,dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, acyclodextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin,hydroxyethyl-β-cyclodextrin, hydroxypropyl-μ-cyclodextrin,hydroxyethyl-γ-cyclodextrin, hydroxypropyl-γ-cyclodextrin,dihydroxypropyl-μ-cyclodextrin, sulfobutylether-β-cyclodextrin,sulfobutylether-γ-cyclodextrin, glucosyl-α-cyclodextrin,glucosyl-β-cyclodextrin, diglucosyl-μ-cyclodextrin,maltosyl-α-cyclodextrin, maltosyl-μ-cyclodextrin,maltosyl-γ-cyclodextrin, maltotriosyl-β-cyclodextrin,maltotriosyl-γ-cyclodextrin, dimaltosyl-β-cyclodextrin,methyl-μ-cyclodextrin, a carboxyalkyl thioether, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, a vinylacetate copolymer, vinyl pyrrolidone, sodium lauryl sulfate, dioctylsodium sulfosuccinate, or any combination thereof.

The pharmaceutical compositions can be formulated by techniques known tothe person skilled in the art, such as the techniques published in“Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press,22^(nd) edition. The pharmaceutical compositions can be formulated asdosage forms for oral, parenteral, such as intramuscular, intravenous,subcutaneous, intradermal, intraarterial, intracardial, rectal, nasal,topical, aerosol or vaginal administration. Dosage forms for oraladministration include coated and uncoated tablets, soft gelatincapsules, hard gelatin capsules, lozenges, troches, solutions,emulsions, suspensions, syrups, elixirs, powders and granules forreconstitution, dispersible powders and granules, medicated gums,chewing tablets and effervescent tablets. Dosage forms for parenteraladministration include solutions, emulsions, suspensions, dispersionsand powders and granules for reconstitution. Emulsions are a preferreddosage form for parenteral administration. Dosage forms for rectal andvaginal administration include suppositories and ovula. Dosage forms fornasal administration can be administered via inhalation andinsufflation, for example by a metered inhaler. Dosage forms for topicaladministration include creams, gels, ointments, salves, patches andtransdermal delivery systems.

The compounds provided herein, particularly the compounds of formula(I), or the above described pharmaceutical compositions comprising sucha compound may be administered to a subject by any convenient route ofadministration, whether systemically/peripherally or at the site ofdesired action, including but not limited to one or more of: oral (e.g.,as a tablet, capsule, or as an ingestible solution), topical (e.g.,transdermal, intranasal, ocular, buccal, and sublingual), parenteral(e.g., using injection techniques or infusion techniques, and including,for example, by injection, e.g., subcutaneous, intradermal,intramuscular, intravenous, intraarterial, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, orintrasternal by, e.g., implant of a depot, for example, subcutaneouslyor intramuscularly), pulmonary (e.g., by inhalation or insufflationtherapy using, e.g., an aerosol, e.g., through mouth or nose),gastrointestinal, intrauterine, intraocular, subcutaneous, ophthalmic(including intravitreal or intracameral), rectal, or vaginaladministration.

If said compounds or pharmaceutical compositions are administeredparenterally, then examples of such administration include one or moreof: intravenously, intraarterially, intraperitoneally, intrathecally,intraventricularly, intraurethrally, intrasternally, intracardially,intracranially, intramuscularly or subcutaneously administering thecompounds or pharmaceutical compositions, and/or by using infusiontechniques. For parenteral administration, the compounds are best usedin the form of a sterile aqueous solution which may contain othersubstances, for example, enough salts or glucose to make the solutionisotonic with blood. The aqueous solutions should be suitably buffered(preferably to a pH of from 3 to 9), if necessary. The preparation ofsuitable parenteral formulations under sterile conditions is readilyaccomplished by standard pharmaceutical techniques well known to thoseskilled in the art.

Said compounds or pharmaceutical compositions can also be administeredorally in the form of tablets, capsules, ovules, elixirs, solutions orsuspensions, which may contain flavoring or coloring agents, forimmediate-, delayed-, modified-, sustained-, pulsed- orcontrolled-release applications.

The tablets may contain excipients such as microcrystalline cellulose,lactose, sodium citrate, calcium carbonate, dibasic calcium phosphateand glycine, disintegrants such as starch (preferably corn, potato ortapioca starch), sodium starch glycolate, croscarmellose sodium andcertain complex silicates, and granulation binders such aspolyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, stearic acid, glycerylbehenate and talc may be included. Solid compositions of a similar typemay also be employed as fillers in gelatin capsules. Preferredexcipients in this regard include lactose, starch, a cellulose, or highmolecular weight polyethylene glycols. For aqueous suspensions and/orelixirs, the agent may be combined with various sweetening or flavoringagents, coloring matter or dyes, with emulsifying and/or suspendingagents and with diluents such as water, ethanol, propylene glycol andglycerin, and combinations thereof.

Alternatively, said compounds or pharmaceutical compositions can beadministered in the form of a suppository or pessary, or may be appliedtopically in the form of a gel, hydrogel, lotion, solution, cream,ointment or dusting powder. The compounds of the present invention mayalso be dermally or transdermally administered, for example, by the useof a skin patch.

Said compounds or pharmaceutical compositions may also be administeredby sustained release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or microcapsules. Sustained-releasematrices include, e.g., polylactides (see, e.g., U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate(Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly(2-hydroxyethylmethacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277(1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinylacetate (R. Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid(EP133988). Sustained-release pharmaceutical compositions also includeliposomally entrapped compounds. Liposomes containing a compound of thepresent invention can be prepared by methods known in the art, such as,e.g., the methods described in any one of: DE3218121; Epstein et al.,Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc.Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP0052322; EP0036676;EP088046; EP0143949; EP0142641; JP 83-118008; U.S. Pat. Nos. 4,485,045;4,544,545; and EP0102324.

Said compounds or pharmaceutical compositions may also be administeredby the pulmonary route, rectal routes, or the ocular route. Forophthalmic use, they can be formulated as micronized suspensions inisotonic, pH adjusted, sterile saline, or, preferably, as solutions inisotonic, pH adjusted, sterile saline, optionally in combination with apreservative such as a benzalkonium chloride. Alternatively, they may beformulated in an ointment such as petrolatum.

It is also envisaged to prepare dry powder formulations of the compoundsprovided herein, particularly the compounds of formula (I), forpulmonary administration, particularly inhalation. Such dry powders maybe prepared by spray drying under conditions which result in asubstantially amorphous glassy or a substantially crystalline bioactivepowder. Accordingly, dry powders of the compounds of the presentinvention can be made according to the emulsification/spray dryingprocess disclosed in WO 99/16419 or WO 01/85136. Spray drying ofsolution formulations of the compounds of the invention can be carriedout, e.g., as described generally in the “Spray Drying Handbook”, 5thed., K. Masters, John Wiley & Sons, Inc., NY (1991), in WO 97/41833, orin WO 03/053411.

For topical application to the skin, said compounds or pharmaceuticalcompositions can be formulated as a suitable ointment containing theactive compound suspended or dissolved in, for example, a mixture withone or more of the following: mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, emulsifying wax and water. Alternatively,they can be formulated as a suitable lotion or cream, suspended ordissolved in, for example, a mixture of one or more of the following:mineral oil, sorbitan monostearate, a polyethylene glycol, liquidparaffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzylalcohol and water.

The present invention thus relates to the compounds or thepharmaceutical compositions provided herein, wherein the correspondingcompound or pharmaceutical composition is to be administered by any oneof: an oral route; topical route, including by transdermal, intranasal,ocular, buccal, or sublingual route; parenteral route using injectiontechniques or infusion techniques, including by subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intracardiac,intrathecal, intraspinal, intracapsular, subcapsular, intraorbital,intraperitoneal, intratracheal, subcuticular, intraarticular,subarachnoid, intrasternal, intraventricular, intraurethral, orintracranial route; pulmonary route, including by inhalation orinsufflation therapy; gastrointestinal route; intrauterine route;intraocular route; subcutaneous route; ophthalmic route, including byintravitreal, or intracameral route; rectal route; or vaginal route.Particularly preferred routes of administration are oral administrationor parenteral administration.

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject. The specific dose level andfrequency of dosage for any particular individual subject may be variedand will depend upon a variety of factors including the activity of thespecific compound employed, the metabolic stability and length of actionof that compound, the age, body weight, general health, sex, diet, modeand time of administration, rate of excretion, drug combination, theseverity of the particular condition, and the individual subjectundergoing therapy.

A proposed, yet non-limiting dose of the compounds according to theinvention for oral administration to a human (of approximately 70 kgbody weight) may be 0.05 to 8000 mg, preferably 0.1 mg to 4000 mg, ofthe active ingredient per unit dose. The unit dose may be administered,e.g., 1 to 3 times per day. The unit dose may also be administered 1 to7 times per week, e.g., with not more than one administration per day. Afurther exemplary dose of the compounds of formula (I) for oraladministration to a human is 50 to 200 mg/kg bodyweight/day,particularly 100 mg/kg/day. It will be appreciated that it may benecessary to make routine variations to the dosage depending on the ageand weight of the patient/subject as well as the severity of thecondition to be treated. The precise dose and also the route ofadministration will ultimately be at the discretion of the attendantphysician or veterinarian.

The compounds provided herein, particularly the compound of formula (I),or a pharmaceutical composition comprising such a compound can beadministered in monotherapy (e.g., without concomitantly administeringany further therapeutic agents, or without concomitantly administeringany further therapeutic agents against the same disease that is to betreated or prevented with the compound of formula (I)). However, thecompound of formula (I) or a pharmaceutical composition comprising thecompound of formula (I) can also be administered in combination with oneor more further therapeutic agents. If the compound of formula (I) isused in combination with a second therapeutic agent active against thesame disease or condition, the dose of each compound may differ fromthat when the corresponding compound is used alone, in particular, alower dose of each compound may be used. The combination of the compoundof formula (I) with one or more further therapeutic agents (such as,e.g., a BRD4 inhibitor, preferably a direct BRD4 inhibitor) may comprisethe simultaneous/concomitant administration of the compound of formula(I) and the further therapeutic agent(s) (either in a singlepharmaceutical formulation or in separate pharmaceutical formulations),or the sequential/separate administration of the compound of formula (I)and the further therapeutic agent(s). If administration is sequential,either the compound of formula (I) according to the invention or the oneor more further therapeutic agents may be administered first. Ifadministration is simultaneous, the one or more further therapeuticagents may be included in the same pharmaceutical formulation as thecompound of formula (I), or they may be administered in one or moredifferent (separate) pharmaceutical formulations.

Preferably, the one or more further therapeutic agents to beadministered in combination with a compound of the present invention areanticancer drugs. The anticancer drug(s) to be administered incombination with a compound of formula (I) according to the inventionmay, e.g., be selected from: a tumor angiogenesis inhibitor (e.g., aprotease inhibitor, an epidermal growth factor receptor kinaseinhibitor, or a vascular endothelial growth factor receptor kinaseinhibitor); a cytotoxic drug (e.g., an antimetabolite, such as purineand pyrimidine analog antimetabolites); an antimitotic agent (e.g., amicrotubule stabilizing drug or an antimitotic alkaloid); a platinumcoordination complex; an anti-tumor antibiotic; an alkylating agent(e.g., a nitrogen mustard or a nitrosourea); an endocrine agent (e.g.,an adrenocorticosteroid, an androgen, an anti-androgen, an estrogen, ananti-estrogen, an aromatase inhibitor, a gonadotropin-releasing hormoneagonist, or a somatostatin analog); or a compound that targets an enzymeor receptor that is overexpressed and/or otherwise involved in aspecific metabolic pathway that is misregulated in the tumor cell (e.g.,ATP and GTP phosphodiesterase inhibitors, histone deacetylaseinhibitors, protein kinase inhibitors (such as serine, threonine andtyrosine kinase inhibitors, e.g., Abelson protein tyrosine kinaseinhibitors) and the various growth factors, their receptors andcorresponding kinase inhibitors (such as epidermal growth factorreceptor kinase inhibitors, vascular endothelial growth factor receptorkinase inhibitors, fibroblast growth factor inhibitors, insulin-likegrowth factor receptor inhibitors and platelet-derived growth factorreceptor kinase inhibitors)); methionine, aminopeptidase inhibitors,proteasome inhibitors, cyclooxygenase inhibitors (e.g., cyclooxygenase-1or cyclooxygenase-2 inhibitors), topoisomerase inhibitors (e.g.,topoisomerase I inhibitors or topoisomerase II inhibitors), poly ADPribose polymerase inhibitors (PARP inhibitors), and epidermal growthfactor receptor (EGFR) inhibitors/antagonists.

An alkylating agent which can be used as an anticancer drug incombination with a compound of the present invention may be, forexample, a nitrogen mustard (such as cyclophosphamide, mechlorethamine(chlormethine), uramustine, melphalan, chlorambucil, ifosfamide,bendamustine, or trofosfamide), a nitrosourea (such as carmustine,streptozocin, fotemustine, lomustine, nimustine, prednimustine,ranimustine, or semustine), an alkyl sulfonate (such as busulfan,mannosulfan, or treosulfan), an aziridine (such as hexamethylmelamine(altretamine), triethylenemelamine, ThioTEPA(N,N′N′-triethylenethiophosphoramide), carboquone, or triaziquone), ahydrazine (such as procarbazine), a triazene (such as dacarbazine), oran imidazotetrazine (such as temozolomide).

A platinum coordination complex which can be used as an anticancer drugin combination with a compound of the present invention may be, forexample, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin,or triplatin tetranitrate.

A cytotoxic drug which can be used as an anticancer drug in combinationwith a compound of the present invention may be, for example, anantimetabolite, including folic acid analogue antimetabolites (such asaminopterin, methotrexate, pemetrexed, or raltitrexed), purine analogueantimetabolites (such as cladribine, clofarabine, fludarabine,6-mercaptopurine (including its prodrug form azathioprine), pentostatin,or 6-thioguanine), and pyrimidine analogue antimetabolites (such ascytarabine, decitabine, 5-fluorouracil (including its prodrug formscapecitabine and tegafur), floxuridine, gemcitabine, enocitabine, orsapacitabine).

An antimitotic agent which can be used as an anticancer drug incombination with a compound of the present invention may be, forexample, a taxane (such as docetaxel, larotaxel, ortataxel,paclitaxel/taxol, tesetaxel, or nab-paclitaxel (e.g., Abraxane®)), aVinca alkaloid (such as vinblastine, vincristine, vinflunine, vindesine,or vinorelbine), an epothilone (such as epothilone A, epothilone B,epothilone C, epothilone D, epothilone E, or epothilone F) or anepothilone B analogue (such as ixabepilone/azaepothilone B).

An anti-tumor antibiotic which can be used as an anticancer drug incombination with a compound of the present invention may be, forexample, an anthracycline (such as aclarubicin, daunorubicin,doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin,or zorubicin), an anthracenedione (such as mitoxantrone, or pixantrone)or an anti-tumor antibiotic isolated from Streptomyces (such asactinomycin (including actinomycin D), bleomycin, mitomycin (includingmitomycin C), or plicamycin).

A tyrosine kinase inhibitor which can be used as an anticancer drug incombination with a compound of the present invention may be, forexample, axitinib, bosutinib, cediranib, dasatinib, erlotinib,gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib,sorafenib, sunitinib, axitinib, nintedanib, ponatinib, or vandetanib.

A topoisomerase inhibitor which can be used as an anticancer drug incombination with a compound of the present invention may be, forexample, a topoisomerase I inhibitor (such as irinotecan, topotecan,camptothecin, belotecan, rubitecan, or lamellarin D) or a topoisomeraseII inhibitor (such as amsacrine, etoposide, etoposide phosphate,teniposide, or doxorubicin).

A PARP inhibitor which can be used as an anticancer drug in combinationwith a compound of the present invention may be, for example, BMN-673,olaparib, rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, or3-aminobenzamide.

An EGFR inhibitor/antagonist which can be used as an anticancer drug incombination with a compound of the present invention may be, forexample, gefitinib, erlotinib, lapatinib, afatinib, neratinib, ABT-414,dacomitinib, AV-412, PD 153035, vandetanib, PKI-166, pelitinib,canertinib, icotinib, poziotinib, BMS-690514, CUDC-101, AP26113, XL647,cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.

Further anticancer drugs may also be used in combination with a compoundof the present invention. The anticancer drugs may comprise biologicalor chemical molecules, like TNF-related apoptosis-inducing ligand(TRAIL), tamoxifen, amsacrine, bexarotene, estramustine, irofulven,trabectedin, cetuximab, panitumumab, tositumomab, alemtuzumab,bevacizumab, edrecolomab, gemtuzumab, alvocidib, seliciclib,aminolevulinic acid, methyl aminolevulinate, efaproxiral, porfimersodium, talaporfin, temoporfin, verteporfin, alitretinoin, tretinoin,anagrelide, arsenic trioxide, atrasentan, bortezomib, carmofur,celecoxib, demecolcine, elesclomol, elsamitrucin, etoglucid, lonidamine,lucanthone, masoprocol, mitobronitol, mitoguazone, mitotane, oblimersen,omacetaxine, sitimagene, ceradenovec, tegafur, testolactone,tiazofurine, tipifarnib, vorinostat, or iniparib.

Also biological drugs, like antibodies, antibody fragments, antibodyconstructs (for example, single-chain constructs), and/or modifiedantibodies (like CDR-grafted antibodies, humanized antibodies, “fullhumanized” antibodies, etc.) directed against cancer or tumormarkers/factors/cytokines involved in proliferative diseases can beemployed in cotherapy approaches with the compounds of the invention.Examples of such biological molecules are anti-HER2 antibodies (e.g.trastuzumab, Herceptin®), anti-CD20 antibodies (e.g. Rituximab,Rituxan®, MabThera®, Reditux®), anti-CD19/CD3 constructs (see, e.g.,EP1071752) and anti-TNF antibodies (see, e.g., Taylor P C. Antibodytherapy for rheumatoid arthritis. Curr Opin Pharmacol. 2003.3(3):323-328). Further antibodies, antibody fragments, antibodyconstructs and/or modified antibodies to be used in cotherapy approacheswith the compounds of the invention can be found, e.g., in: Taylor P C.Curr Opin Pharmacol. 2003. 3(3):323-328; or Roxana A. Maedica. 2006.1(1):63-65.

An anticancer drug which can be used in combination with a compound ofthe present invention may, in particular, be an immunooncologytherapeutic (such as an antibody (e.g., a monoclonal antibody or apolyclonal antibody), an antibody fragment, an antibody construct (e.g.,a single-chain construct), or a modified antibody (e.g., a CDR-graftedantibody, a humanized antibody, or a “full humanized” antibody)targeting any one of CTLA-4, PD-1/PD-L1, TIM3, LAG3, OX4, CSF1R, IDO, orCD40. Such immunooncology therapeutics include, e.g., an anti-CTLA-4antibody (particularly an antagonistic or pathway-blocking anti-CTLA-4antibody; e.g., ipilimumab or tremelimumab), an anti-PD-1 antibody(particularly an antagonistic or pathway-blocking anti-PD-1 antibody;e.g., nivolumab (BMS-936558), pembrolizumab (MK-3475), pidilizumab(CT-011), AMP-224, or APE02058), an anti-PD-L1 antibody (particularly apathway-blocking anti-PD-L1 antibody; e.g., BMS-936559, MEDI4736,MPDL3280A (RG7446), MDX-1105, or MEDI6469), an anti-TIM3 antibody(particularly a pathway-blocking anti-TIM3 antibody), an anti-LAGSantibody (particularly an antagonistic or pathway-blocking anti-LAG3antibody; e.g., BMS-986016, IMP701, or IMP731), an anti-OX4 antibody(particularly an agonistic anti-OX4 antibody; e.g., MED10562), ananti-CSF1R antibody (particularly a pathway-blocking anti-CSF1Rantibody; e.g., IMC-CS4 or RG7155), an anti-IDO antibody (particularly apathway-blocking anti-IDO antibody), or an anti-CD40 antibody(particularly an agonistic anti-CD40 antibody; e.g., CP-870,893 or ChiLob 7/4). Further immunooncology therapeutics are known in the art andare described, e.g., in: Kyi C et al., FEBS Lett, 2014, 588(2):368-76;Intlekofer A M et al., J Leukoc Biol, 2013, 94(1):25-39; Callahan M K etal., J Leukoc Biol, 2013, 94(1):41-53; Ngiow S F et al., Cancer Res,2011, 71(21):6567-71; and Blattman J N et al., Science, 2004,305(5681):200-5.

A BRD4 inhibitor (preferably a direct BRD4 inhibitor), such as CeMMEC2,may also be used as a further therapeutic agent in combination with thecompound of formula (I).

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation. The individual componentsof such combinations may be administered either sequentially orsimultaneously/concomitantly in separate or combined pharmaceuticalformulations by any convenient route. When administration is sequential,either the compound of the present invention (particularly the compoundof formula (I) or a pharmaceutically acceptable salt, solvate or prodrugthereof) or the further therapeutic agent(s) may be administered first.When administration is simultaneous, the combination may be administeredeither in the same pharmaceutical composition or in differentpharmaceutical compositions. When combined in the same formulation, itwill be appreciated that the two or more compounds must be stable andcompatible with each other and the other components of the formulation.When formulated separately, they may be provided in any convenientformulation.

The compounds provided herein, particularly the compounds of formula(I), can also be administered in combination with physical therapy, suchas radiotherapy. Radiotherapy may commence before, after, orsimultaneously with administration of the compounds of the invention.For example, radiotherapy may commence 1-10 minutes, 1-10 hours or 24-72hours after administration of the compounds. Yet, these time frames arenot to be construed as limiting. The subject is exposed to radiation,preferably gamma radiation, whereby the radiation may be provided in asingle dose or in multiple doses that are administered over severalhours, days and/or weeks. Gamma radiation may be delivered according tostandard radiotherapeutic protocols using standard dosages and regimens.

The present invention thus relates to a compound of formula (I) or apharmaceutically acceptable salt, solvate, or prodrug thereof, or apharmaceutical composition comprising any of the aforementioned entitiesin combination with a pharmaceutically acceptable excipient, for use inthe treatment or prevention of cancer, wherein the compound or thepharmaceutical composition is to be administered in combination with oneor more anticancer drugs and/or in combination with radiotherapy.

Yet, the compounds of formula (I) can also be used in monotherapy,particularly in the monotherapeutic treatment or prevention of cancer(i.e., without administering any other anticancer agents until thetreatment with the compound(s) of formula (I) is terminated).Accordingly, the invention also relates to a compound of formula (I) ora pharmaceutically acceptable salt, solvate, or prodrug thereof, or apharmaceutical composition comprising any of the aforementioned entitiesin combination with a pharmaceutically acceptable excipient, for use inthe monotherapeutic treatment or prevention of cancer.

The subject or patient to be treated in accordance with the presentinvention may be an animal (e.g., a non-human animal), a vertebrateanimal, a mammal, a rodent (e.g., a guinea pig, a hamster, a rat, or amouse), a canine (e.g., a dog), a feline (e.g., a cat), a porcine (e.g.,a pig), an equine (e.g., a horse), a primate or a simian (e.g., a monkeyor an ape, such as a marmoset, a baboon, a gorilla, a chimpanzee, anorangutan, or a gibbon), or a human. In accordance with the presentinvention, it is envisaged that animals are to be treated which areeconomically, agronomically or scientifically important. Scientificallyimportant organisms include, but are not limited to, mice, rats, andrabbits. Lower organisms such as, e.g., fruit flies like Drosophilamelagonaster and nematodes like Caenorhabditis elegans may also be usedin scientific approaches. Non-limiting examples of agronomicallyimportant animals are sheep, cattle and pigs, while, for example, catsand dogs may be considered as economically important animals.Preferably, the subject/patient is a mammal. More preferably, thesubject/patient is a human or a non-human mammal (such as, e.g., aguinea pig, a hamster, a rat, a mouse, a rabbit, a dog, a cat, a horse,a monkey, an ape, a marmoset, a baboon, a gorilla, a chimpanzee, anorangutan, a gibbon, a sheep, cattle, or a pig). Most preferably, thesubject/patient is a human.

The term “treatment” of a disorder or disease as used herein (e.g.,“treatment” of cancer) is well known in the art. “Treatment” of adisorder or disease implies that a disorder or disease is suspected orhas been diagnosed in a patient/subject. A patient/subject suspected ofsuffering from a disorder or disease typically shows specific clinicaland/or pathological symptoms which a skilled person can easily attributeto a specific pathological condition (i.e., diagnose a disorder ordisease).

The “treatment” of a disorder or disease may, for example, lead to ahalt in the progression of the disorder or disease (e.g., nodeterioration of symptoms) or a delay in the progression of the disorderor disease (in case the halt in progression is of a transient natureonly). The “treatment” of a disorder or disease may also lead to apartial response (e.g., amelioration of symptoms) or complete response(e.g., disappearance of symptoms) of the subject/patient suffering fromthe disorder or disease. Accordingly, the “treatment” of a disorder ordisease may also refer to an amelioration of the disorder or disease,which may, e.g., lead to a halt in the progression of the disorder ordisease or a delay in the progression of the disorder or disease. Such apartial or complete response may be followed by a relapse. It is to beunderstood that a subject/patient may experience a broad range ofresponses to a treatment (such as the exemplary responses as describedherein above). The treatment of a disorder or disease may, inter alia,comprise curative treatment (preferably leading to a complete responseand eventually to healing of the disorder or disease) and palliativetreatment (including symptomatic relief).

The term “prevention” of a disorder or disease as used herein (e.g.,“prevention” of cancer) is also well known in the art. For example, apatient/subject suspected of being prone to suffer from a disorder ordisease may particularly benefit from a prevention of the disorder ordisease. The subject/patient may have a susceptibility or predispositionfor a disorder or disease, including but not limited to hereditarypredisposition. Such a predisposition can be determined by standardmethods or assays, using, e.g., genetic markers or phenotypicindicators. It is to be understood that a disorder or disease to beprevented in accordance with the present invention has not beendiagnosed or cannot be diagnosed in the patient/subject (for example,the patient/subject does not show any clinical or pathologicalsymptoms). Thus, the term “prevention” comprises the use of a compoundof the present invention before any clinical and/or pathologicalsymptoms are diagnosed or determined or can be diagnosed or determinedby the attending physician.

It is to be understood that the present invention specifically relatesto each and every combination of features and embodiments describedherein, including any combination of general and/or preferredfeatures/embodiments. In particular, the invention specifically relatesto each combination of meanings (including general and/or preferredmeanings) for the various groups and variables comprised in formula (I).

In this specification, a number of documents including patentapplications, scientific literature and manufacturers' manuals arecited. The disclosure of these documents, while not considered relevantfor the patentability of this invention, is herewith incorporated byreference in its entirety. More specifically, all referenced documentsare incorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

The invention is also described by the following illustrative figures.The appended figures show:

FIG. 1: Generation of a reporter cell line for the inhibition of BRD4

(a) Graphic representation of the experimental approach. WT-KBM7 cellswere treated with 0.5 μM (S)-JQ1 for 18 hours and then infected with theLZRS-RFP-ires-ZEO retroviral vector. RFP-positive cells were sorted inpresence of 0.5 μM (S)-JQ1. (S)-JQ1 was removed from the media and theRFP negative population was sorted into single cell clones. All theclones were treated several times with (S)-JQ1 to check whether thetreatment was stably inducing RFP expression. (b) Sorting panelsrepresenting the WT-KBM7 population (not infected), the infected andsorted population (red square: RFP-positive and sorted cells), and thedouble sorted population (black square: RFP negative and double sortedcells). (c) Representative FACS panels of REDS3 cells treated with 0.5μM (R)-JQ1 or (S)-JQ1 for 18 hours; an equal volume of DMSO was used ascontrol. At least three biological replicates were done for eachexperimental condition. (d) Quantification of RFP-positive cells byFACS, following downregulation of the indicated bromodomain proteins inREDS3 cells (BRDW1 and BRD1 were used as negative controls); threebiological replicates were done for each experimental condition and atleast 30,000 cells were analyzed each time (mean±STD). (e)Representative images of REDS3 cells downregulated for BRD3 or BRD4(hairpin number 2 for both of these bromodomains).

FIG. 2: Transcriptional repression of BRD4 target genes inducesupregulation of flanking regions

(a) Representative pictures of the FISH assay done in REDS3 cells. RFPprobe (purple dots) stains the RFP insertion; Hoechst (blue signal)stains the nucleus. Yellow dashed lines mark nuclear perimeter. Scalebar is 10 μM. (b) Quantification of RFP probe localization with respectto the nuclear membrane. Near and far indicate the distance between theRFP FISH probe and the nuclear membrane (0<near<2 μM; far>2 μM);duplicates were performed and at least 80 cells were counted for eachexperimental condition (mean±STD). (c) Representation of the RFP locus.RFP (red arrow) is inserted in the sense direction at 12 chromosome(chr12:131,323,912-131,324,450) between STX2 (reverse direction, cyanarrow) and RAN (sense direction, cyan arrow); primers used in d areindicated in the representation (WT genome: green lines; REDS3 genome:purple lines). The region between STX2 and RAN is enhancer-rich (lightorange dashed line), while the region downstream of STX2 isheterochromatic (gray dashed loops). In the box: representation ofENCODE/Broad institute chromatin state segmentation and Chip-seq (H3K4ml, H3K4m3, H3K27ac, H3K27m3 and H3H36m3) data (K-562 cell line). (d)PCR of WT and REDS3 DNA using specific primers for the locus of the RFPinsertion; primers amplifying the insulin promoter (Ins_P) have beenused as control. (e) Volcano plot representing gene expression changesin KBM7 cells upon treatment with 1 μM (S)-JQ1 for 24 hours, compared toDMSO treatment (RNAseq data analysis; grey dots, not significant(qvalue>0.05)/red dots, significant (qvalue<0.05)). (f) DAVID functionalannotation analysis of WT-KBM7 (S)-JQ1-upregulated genes. (g) RT-PCRshowing STX2 and RAN fold change upon treatment with (S)-JQ1 1 μM for 24hours in KBM7 cells. Values are normalized to actin expression and DMSOtreated cells. Three biological replicates were done for each condition(mean±STD).

FIG. 3: Screening for functional BRD4 inhibitors

(a) Heat map showing the increase of RFP-positive nuclei in REDS3 clonetreated with the showed compounds at 1, 5 and 10 μM for 24 hours(triplicates, % of control, DMSO is used as negative control and (S)-JQ120 μM as positive control). (b) Scatter plot representing the hitdistribution from the last part of the screening (validation part). Thevariable RED was calculated as the product of the number of red cellsmultiplied by the median red intensity. Autofluorescent compoundsincrease RED in WT-KBM7 cells, whereas hit compounds act only in RED3cells. (c) Percentage of c-MYC expression in WT-KBM7 treated with theselected compounds, assessed by RT-PCR. 1, 10 and 20 μM of each compoundwere used to treat cells for 24 hours; DMSO was used as negative controland (S)-JQ1 as positive control. Three biological replicates wereperformed (mean±STD). (d) Chemical structures of (S)-JQ1, CeMMEC1 andCeMMEC2. (e) Quantification of cells in S-phase by staining the nucleiwith PI and analyzing DNA content by FACS. THP1 cells were treated withDMSO or the indicated concentrations of (S)-JQ1, CeMMEC1 or CeMMEC2 for48 hours. Three different biological replicates were performed and30,000 cells were analysed each time (mean±STD). (f) Quantification ofAnnexinV positive cells from immunofluorescence images. Cells weretreated with DMSO or the indicated concentrations of (S)-JQ1, CeMMEC1 orCeMMEC2 for 72 hours. At least 3 biological replicates were performedand more than 1,500 cells were quantified for each point (mean±STD).

FIG. 4: Molecular and cellular characterization of CeMMEC1 and CeMMEC2

(a) AlphaLISA assays for the first (black columns, BD1) and the second(grey columns, BD2) bromodomains of BRD4, incubated with CeMMEC1 andCeMMEC2. (S)-JQ1 and RVX-208 were used as positive controls. The assaywas done in duplicate (mean±STD); all compounds were used at 10 μM. (b)AlphaLISA dose response (12 dilutions, from 20 to 0.02 μM) for fulllength BRD4 (GST-tagged) incubated with (S)-JQ1 or CeMMEC2. (c)BromoScan profile for CeMMEC1 (red bubbles) and CeMMEC2 (pink bubbles)(10 uM). Bubbles indicate the percentage of inhibition of the binding ofthe analyzed bromodomains to an acetylated substrate. (d) RepresentativeWestern Blots showing knock down levels of the indicated bromodomaindownregulated using two different shRNAs (shRNAC=control_sh;shRNA1=hairpin n.1; shRNA2=hairpin n.2.) (e) RFP-positive REDS3 cellsquantification upon downregulation of the indicated bromodomains fromlive cell imaging pictures. Three replicates were perfromed and morethan 1,500 cells were quantified for each point (% of control, positivecontrol is shControl treated with DMSO, positive control is shControltreated with (S)-JQ1; mean±STD). (f) RFP-positive cell fold changequantified from live cell imaging pictures of REDS3 clone treated withthe indicated compounds at the displayed concentrations for 24 hours(DMSO normalized; duplicates, at least 1500 cells were quantified ineach replicate). (g) BromoKdELECT assay for CeMMEC1 against TAF1 (2).(h) Docking of CeMMEC2 to the first bromodomain of BRD4. (i) Docking ofCeMMEC1 to the second bromodomain of TAF1. 3D model (top panel) and 2Dligand interaction diagram (bottom panel). Red dots in the 3D modelindicate water molecules.

FIG. 5: TAF1 synergizes with BRD4 to mediate transcriptional control

(a) Fold change c-MYC expression assessed by RT-PCR in WT-KBM7 withknockdown of TAF1 or BRD4 (compared to control cells); three biologicalreplicates were performed (mean±STD). (b, c) CellTiterGlo assay ofcontrol and TAF1 downregulated WT-KBM7 treated with the indicatedconcentrations of (b) (S)-JQ1 or (c) CeMMEC2 (each point performed atleast in triplicate, an equal amount of DMSO was added as control). (d)Chemical structures of the indicated CeMMEC1 analogs. (e) RFP-positivecell fold change quantified from live cell imaging pictures of REDS3clone treated with the indicated compounds at the displayedconcentrations for 24 hours (DMSO normalized; duplicates, at least 1500cells were quantified in each replicate). (f) Matrix displaying foldchange of REDS3 RFP-positive cells treated with the indicatedconcentrations of (S)-JQ1, CeMMEC1, analog 29, analog 30, analog 32 andanalog 35 alone or in combination (each point at least in triplicate).(g) Matrix displaying cell viability reduction of H23 cells treated withthe indicated concentrations of (S)-JQ1, CeMMEC1, analog 29, analog 30,analog 32 and analog 35 alone or in combination (each point done atleast in duplicate, an equal amount of DMSO was added as control).Statistics (Student's t-test, two tails): * indicates 0.05<pvalue<0.01;** indicates 0.01<pvalue<0.001; *** indicates pvalue<0.001.

FIG. 6: Characterization of RED3 cells

(a) Western Blot analysis showing c-MYC downregulation in WT-KBM7treated with 0.5 μM (S)-JQ1 for 18 hours; an equal volume of DMSO wasused as control. (b) S-phase quantification from cell cycle profilesevaluated by PI-staining and DNA content analysis by FACS. WT-KBM7 cellswere treated with 0.5 μM of (S)-JQ1 or (R)-JQ1 for 24 hours; an equalamount of DMSO was added as control. Four biological replicates havebeen done (mean). (c) Examples of live cell imaging pictures of REDS1,REDS2 and REDS3 cells treated with 0.5 μM of (S)-JQ1 or (R)-JQ1; anequal amount of DMSO was added as control. (d) RFP and (e) zeocinexpression performed by RT-PCR in REDS3 cells treated with 0.5 μM(S)-JQ1 or (R)-JQ1 for 18 hours; an equal volume of DMSO was used ascontrol. Three biological replicates were performed for eachexperimental condition (mean±STD). (f) Left panel: cell cycle profilesof REDS3 untreated cells or REDS3 cells treated for 20 hours withthymidine (2 mM), R03306 (9 μM) or nocodazole (1 μM). Nuclei werestained with PI and DNA content quantified by FACS; representative cellcycle profiles from one of the two experiments done (30,000 cells wereFACS analyzed in each experiment). Right panel: examples of live cellimaging pictures of REDS3 cells treated as above and with 0.5 μM of(S)-JQ1; an equal volume of DMSO was used as control. Representativepictures from one of the three replicates done. (g) Quantification ofRFP-positive cells by live cell imaging pictures of REDS3 cells treatedwith PMA (200 nM), PHA (5ug/ml), (S)-JQ1 (1 μM) or a combination of PMAor PHA with (S)-JQ1 for 24 hours. An equal amount of DMSO was added ascontrol; three replicates were done and at least 1,500 cells werequantified. (h) BRD3, BRD4 and RFP expression assessed by RT-PCR in BRD3or BRD4 downregulated REDS3 cells; three biological replicates were donefor each experimental condition (mean±STD).

FIG. 7: Characterization of screening hits

(a) Pipeline for the selection of validated hits. Compound library:collection of 89,355 small molecules. DR: Dose Response; TC: TimeCourse. (b) Representation of the criteria used for the selection of thepositive control concentration ((S)-JQ1 0.5 μM) applied in the screeningin terms of RFP fluorescence increase (from live cell imaging picturesquantification, duplicates) and (c) its associated Z-Factor. (d) Exampleof live cell imaging pictures of REDS3 cells treated with 10 μM of(S)-JQ1, CeMMEC1 or CeMMEC2 for 24 hours. An equal volume of DMSO wasused as control. (e) Quantification of RFP-positive nuclei of REDS3cells treated with (S)-JQ1, CeMMEC1 and CeMMEC2 at 0.5, 1, 2.5, 5, 10and 20 μM from live cell imaging pictures. An equal amount of DMSO wasadded as control; three replicates were done and at least 1,500 cellswere quantified in each experiment (mean±STD). (f) RNA-seq analysis:Venn diagrams indicating the number of up- and down-regulated genes inWT-KBM7 cells treated with (S)-JQ1 1 μM (grey), CeMMEC1 10 μM (red) andCeMMEC2 10 μM (pink) for 24 hours, and the overlapping of these groups.(g) Scatter plot representing the correlation of gene expressionvariation in cells treated as in D.

FIG. 8: Characterization of the binding of CeMMEC1 and CeMMEC2 tobromodomain proteins

(a) Dose response (12 dilutions, from 20 to 0.02 μM) AlphaLISA assay forthe first (BD1) and the second (BD2) bromodomain of BRD4 incubated with(S)-JQ1 and CeMMEC2. The assay was done in duplicate (mean±STD); (b)BromoKdELECT assay for CeMMEC1 against TAF1 (2), BRD9, CREBBP and EP300bromodomains (11 dilutions, from 10 to 0.017 μM). (c) not used (d)KinomeScan profile for CeMMEC1 (10 μM). Bubbles indicate the percentageof inhibition of the binding of the analyzed kinase to an immobilizedligand. (e) BromoELECT assay for CeMMEC1 and analogs A1, A2, 05, 10, 13,24, 25, 26, 27, 29, 33 and 39 (10 μM) against BRD4 (1), BRD4 (2), BRD9,CREBBP, EP300 and TAF1 (2). (f, g) Docking poses of analog 30 to TAF1(green) and BRD4 (magenta). (h) Docking of compound 29 to TAF1, nosuitable pose could be identified for BRD4. (i) BromoELECT assay foranalog 29 (10 μM) against BRD4 (1), BRD9, CREBBP, EP300 and TAF1 (2).

FIG. 9: Interaction of BRD4 and TAF1

(a) Fold change RFP expression assessed by RT-PCR in REDS3 cells withshRNA downregulation of TAF1 or BRD4 (compared to control cells); threebiological replicates were done (mean±STD). (b) Flag pull-down usingprotein extracts from HEK293T overexpressing flag-BRD4 (full length) orflag alone (representative images from one of the three experimentsdone). (c) CellTiterGlo assay of control and TAF1 downregulated WT-KBM7treated with the indicated concentrations of CeMMEC1 (each point atleast in triplicate, an equal amount of DMSO was added as control). (d)Matrix displaying cell viability reduction of THP1 treated with theindicated concentrations of (S)-JQ1, CeMMEC1, analog 29, analog 30,analog 32 and analog 35 alone or in combination (each point done atleast in duplicate, equal amount of DMSO was added as control). (e)Differential Volume values as measure of the synergy between (S)-JQ1 andCeMMEC1 or the indicated analogs from the treatments represented in thematrix in d and FIG. 5 g.

FIG. 10: Small molecule screening data

The reference to “supplementary table 1” in this figure refers to FIG.11.

FIG. 11: Summary of bioactivity data of hit compounds Redness:RFP-positive cell fold change after 24 hours treatment. All compounds at10 μM (values are DMSO normalized).

AlphaLISA: POC (percentage of control) of BRD4_BD1 (first bromodomain)binding. All compounds at 10 μM.

“True hit”: identification of alphaLISA false positives (quenchingcompounds) using the true hits kit (PerkinElmer); “Y” means notquenching compounds (true hit=Yes); “N” means quenching compound (truehit=No).

AnnV+: % of AnnexinV positive cells after 48 hours treatment. Allcompounds at 10 μM (values are DMSO normalised).

Cell cycle: cell cycle phenotype after 72 hours treatment. All compoundsat 10 μM.

Myc: % of remaining Myc expression after 24 hours treatment. Allcompounds at 10 μM (values are DMSO normalised).

ND=not determined.

FIG. 12: Bioactivity data of CeMMEC1 and derivatives thereof

Redness (% induction at 10 μM), BRD4_BD1 (% inhibition at 10 μM),BRD4_BD2 (% inhibition at 10 μM), BRD9 (% inhibition at 10 μM), CREBBP(% inhibition at 10 μM), EP300 (% inhibition at 10 μM), and TAF1_BD2 (%inhibition at 10 μM) are shown for CeMMEC1 and derivatives thereof.

The present invention particularly relates to the following items:

-   1. A compound of the following formula (I):

-   -   wherein:        -   ring B is a group having the following structure:

-   -   -   one of the ring atoms X₂ and X₃ is N(R^(X1)), and the other            one of said ring atoms X₂ and X₃ is C(═O);        -   the ring atom X₁ is selected from N(R^(X1)), C(R^(X2)) and            C(═O), and the ring atoms X₄ and X₅ are each independently            selected from N(R^(X1)), C(R^(X3)) and C(═O); wherein at            least one of said ring atoms X₁, X₄, and X₅ is different            from N(R^(X1)) and C(═O); and further wherein if X₃ and X₅            are C(═O), X₄ is N(R^(X1)), and X₁ is C(R^(X2)), then X₂ is            N(H);        -   each            is independently a single bond or a double bond, wherein at            least one of any two adjacent bonds            is a single bond;        -   each R^(X1) is independently selected from hydrogen, C₁₋₅            alkyl, —CO(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-aryl, and —(C₀₋₃            alkylene)-heteroaryl, wherein the aryl comprised in said            —(C₀₋₃ alkylene)-aryl and the heteroaryl comprised in said            —(C₀₋₃ alkylene)-heteroaryl are each optionally substituted            with one or more groups R^(X11).        -   R^(X2) is selected from hydrogen, C₁₋₅ alkyl, C₂₋₅ alkenyl,            C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅            alkyl), —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃            alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl),            —(C₀₋₃alkylene)-N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅ haloalkyl), —(C₀₋₃            alkylene)-O—(C₁₋₅haloalkyl), —(C₀₋₃ alkylene)-CF₃, —(C₀₋₃            alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO,            —(C₀₋₃ alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH,            —(C₀₋₃alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃            alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—N(C₁₋₅            alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl),            —(C₀₋₃alkylene)-SO₂—N(C₁₋₅alkyl)(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-NH—SO₂—(C₁₋₅ alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅            alkyl)-SO₂—(C₁₋₅ alkyl);        -   the two groups R^(X3) are either mutually linked to form,            together with the ring carbon atoms that they are attached            to, a 5- or 6-membered cyclyl group which is optionally            substituted with one or more groups R^(X31), or the two            groups R^(X3) are each independently selected from hydrogen,            C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl),            —O(C₁₋₅ alkylene)-OH, alkylene)-O(C₁₋₅ alkyl), —SH, —S(C₁₋₅            alkyl), —NH₂, —NH(C₁₋₅ alkyl), —N(C₁₋₅ alkyl)(C₁₋₅ alkyl),            halogen, C₁₋₅ haloalkyl, —O—(C₁₋₅ haloalkyl), —CF₃, —CN,            —NO₂, —CHO, —CO—(C₁₋₅ alkyl), —COOH, —CO—O—(C₁₋₅ alkyl),            —O—CO—(C₁₋₅ alkyl), —CO—NH₂, —CO—NH(C₁₋₅ alkyl), —CO—N(C₁₋₅            alkyl)(C₁₋₅ alkyl), —NH—CO—(C₁₋₅ alkyl), —N(C₁₋₅            alkyl)-CO—(C₁₋₅ alkyl), —SO₂—NH₂, —SO₂—NH(C₁₋₅ alkyl),            —SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —NH—SO₂—(C₁₋₅ alkyl), and            —N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl);        -   each R^(X11) is independently selected from C₁₋₅ alkyl, C₂₋₅            alkenyl, C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃            alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O(C₁₋₅            alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅            alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅ haloalkyl), —(C₀₋₃            alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃, —(C₀₋₃            alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO,            —(C₀₋₃ alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH,            —(C₀₋₃ alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—NH₂, —(C₀₋₃            alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—N(C₁₋₅            alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-NH—SO₂—(C₁₋₅ alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅            alkyl)-SO₂—(C₁₋₅ alkyl);        -   each R^(X31) is independently selected from C₁₋₅ alkyl, C₂₋₅            alkenyl, C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃            alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O(C₁₋₅            alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅            alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)(C₁₋₅ alkyl),            —(C₀₋₃alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅ haloalkyl),            —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃,            —(C₀₋₃ alkylene)-CN, —(C₀₋₃alkylene)-NO₂, —(C₀₋₃            alkylene)-CHO, —(C₀₋₃ alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-COOH, —(C₀₋₃ alkylene)-CO—O—(C₁₋₅ alkyl),            —(C₀₋₃alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—NH₂,            —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-NH—CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅            alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SO₂—NH₂, —(C₀₋₃            alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SO₂—N(C₁₋₅            alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅ alkyl),            and —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl);        -   ring B is attached to the remainder of the compound of            formula (I) via the ring carbon atom that is marked with an            asterisk (*) or, if X₄ and X₅ are each C(R^(X3)) and the two            groups R^(X3) are mutually linked to form, together with the            ring carbon atoms that they are attached to, a 5- or            6-membered cyclyl group which is optionally substituted with            one or more groups R^(X31), then ring B may also be attached            to the remainder of the compound of formula (I) via any ring            carbon atom of said 5- or 6-membered cyclyl group;        -   ring A is aryl or heteroaryl, wherein said aryl and said            heteroaryl are each optionally substituted with one or more            groups R^(A);        -   each R^(A) is independently selected from C₁₋₅ alkyl, C₂₋₅            alkenyl, C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃            alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O(C₁₋₅            alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅            alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅ haloalkyl), —(C₀₋₃            alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃, —(C₀₋₃            alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO,            —(C₀₋₃ alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH,            —(C₀₋₃ alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—NH₂, —(C₀₋₃            alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—N(C₁₋₅            alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl),            —(C₀₋₃ alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃            alkylene)-NH—SO₂—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅            alkyl)-SO₂—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-cycloalkyl, —(C₀₋₃            alkylene)-O-cycloalkyl, —(C₀₋₃ alkylene)-O(C₁₋₅            alkylene)-cycloalkyl, —(C₀₋₃ alkylene)-heterocycloalkyl,            —(C₀₋₃ alkylene)-O-heterocycloalkyl, and —(C₀₋₃            alkylene)-O(C₁₋₅ alkylene)-heterocycloalkyl;        -   L is selected from —CO—N(R^(L1))—, —N(R^(L1))—CO—, —CO—O—,            —O—CO—, —C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—,            —C(═S)—N(R^(L1))—, —N(R^(L1))—C(═S)—,            —N(R^(L1))—CO—N(R^(L1))—, —O—CO—N(R^(L1))—,            —N(R^(L1))—CO—O—, —N(R^(L1))—C(═N—R^(L2))—N(R^(L1))—,            —N(R^(L1))—C(═N—R^(L2))—O—, —S—C(═N—R^(L2))—N(R^(L1))—,            —N(R^(L1))—C(═N—R^(L2))—S—, —N(R^(L1))—C(═S)—N(R^(L1))—,            —O—C(═S)—N(R^(L1))—, —N(R^(L1))—C(═S)—O—, —S—CO—N(R^(L1))—,            and —N(R^(L1))—CO—S—;        -   each R^(L1) is independently selected from hydrogen and C₁₋₅            alkyl;        -   each R^(L2) is independently selected from hydrogen, C₁₋₅            alkyl, —CN, and —NO₂;        -   n is 0 or 1; and        -   m is 0 or 1;

    -   or a pharmaceutically acceptable salt, solvate or prodrug        thereof

    -   for use as a medicament.

-   2. The compound for use according to item 1, wherein X₂ is C(═O),    and wherein X₃ is N(R^(X1)).

-   3. The compound for use according to item 1, wherein X₂ is    N(R^(X1)), and wherein X₃ is C(═O).

-   4. The compound for use according to any one of items 1 to 3,    wherein X₁ is C(R^(X2)), and wherein X₄ and X₅ are each C(R^(X3)).

-   5. The compound for use according to item 4, wherein the bond    between the ring atom X₁ and the ring carbon atom which is bound to    the moiety —(CH₂)_(n)-L-(CH₂)_(m)— is a double bond, the bond    between said ring carbon atom which is bound to the moiety    —(CH₂)_(n)-L-(CH₂)_(m)— and the ring atom X₅ is a single bond, and    the bond    between the ring atoms X₄ and X₅ is a double bond.

-   6. The compound for use according to any one of items 1 to 5,    wherein each R^(X1) is independently selected from hydrogen and C₁₋₅    alkyl.

-   7. The compound for use according to any one of items 1 to 6,    wherein each R^(X1) is methyl.

-   8. The compound for use according to any one of items 1 to 7,    wherein R^(X2) is selected from hydrogen, C₁₋₄ alkyl, —OH, —O(C₁₋₄    alkyl), —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)(C₁₋₄ alkyl), halogen,    —CF₃, and —CN.

-   9. The compound for use according to any one of items 1 to 8,    wherein ring B is attached to the remainder of the compound of    formula (I) via the ring carbon atom that is marked with an    asterisk.

-   10. The compound for use according to any one of items 1 to 8,    wherein X₄ and X₅ are each C(R^(X3)) and the two groups R^(X3) are    mutually linked to form, together with the ring carbon atoms that    they are attached to, a 5- or 6-membered cyclyl group, and wherein    ring B is attached to the remainder of the compound of formula (I)    via any ring carbon atom of said 5- or 6-membered cyclyl group.

-   11. The compound for use according to any one of items 1 to 10,    wherein the two groups R^(X3) are mutually linked to form, together    with the ring carbon atoms that they are attached to, a 5- or    6-membered cycloalkyl group, a 5- or 6-membered cycloalkenyl group,    or a phenyl group, wherein said cycloalkyl group, said cycloalkenyl    group and said phenyl group are each optionally substituted with one    or more groups R^(X31)

-   12. The compound for use according to any one of items 1 to 11,    wherein ring A is selected from 1,4-benzodioxanyl, benzoxanyl,    1,3-benzodioxolanyl, benzoxolanyl, 1,5-benzodioxepanyl,    benzodioxepanyl, phenyl, and a 5- or 6-membered monocyclic    heteroaryl, wherein said 1,4-benzodioxanyl, said benzoxanyl, said    1,3-benzodioxolanyl, said benzoxolanyl, said 1,5-benzodioxepanyl,    said benzodioxepanyl, said phenyl, and said heteroaryl are each    optionally substituted with one or more groups R^(A).

-   13. The compound for use according to any one of items 1 to 12,    wherein ring A is selected from 1,4-benzodioxan-6-yl,    1-benzoxan-6-yl, and 4-methoxyphenyl.

-   14. The compound for use according to any one of items 1 to 13,    wherein L is —CO—N(R^(L1))— or —N(R^(L1))—CO—.

-   15. The compound for use according to any one of items 1 to 14,    wherein L is —N(R^(L1))—CO—, wherein said —N(R^(L1))—CO— is bound    via its —N(R^(L1))— group to the moiety —(CH₂)_(n)—, and via its    —CO— group to the moiety —(CH₂)_(m)—

-   16. The compound for use according to any one of items 1 to 14,    wherein L is —CO—N(R^(L1))—, wherein said —CO—N(R^(L1))— is bound    via its —CO— group to the moiety —(CH₂)_(n)—, and via its    —N(R^(L1))— group to the moiety —(CH₂)_(m)—.

-   17. The compound for use according to any one of items 14 to 16,    wherein R^(L1) is hydrogen.

-   18. The compound for use according to any one of items 14 to 17,    wherein n is 0.

-   19. The compound for use according to any one of items 14 to 18,    wherein m is 0.

-   20. The compound for use according to item 1, wherein said compound    is a compound of any one of the following formulae:

-   -   or a pharmaceutically acceptable salt, solvate or prodrug        thereof.

-   21. A pharmaceutical composition comprising a compound as defined in    any one of items 1 to 20 and a pharmaceutically acceptable    excipient.

-   22. A compound as defined in any one of items 1 to 20 or the    pharmaceutical composition of item 21 for use in the treatment or    prevention of cancer.

-   23. Use of a compound as defined in any one of items 1 to 20 in the    preparation of a medicament for the treatment or prevention of    cancer.

-   24. A method of treating or preventing cancer, the method comprising    administering a compound as defined in any one of items 1 to 20 or    the pharmaceutical composition of item 21 to a subject in need    thereof.

-   25. The compound for use according to item 22 or the pharmaceutical    composition for use according to item 22 or the use of item 23 or    the method of item 24, wherein said cancer is selected from prostate    carcinoma, breast cancer, acute myeloid leukemia, multiple myeloma,    glioblastoma, and NUT midline carcinoma.

-   26. The compound for use according to any one of items 1 to 20, 22    or 25 or the pharmaceutical composition for use according to item 22    or 25 or the use of item 23 or 25 or the method of item 24 or 25,    wherein the subject to be treated is a human.

-   27. The compound for use according to any one of items 1 to 20, 22,    25 or 26 or the pharmaceutical composition for use according to item    22, 25 or 26 or the use of item 23, 25 or 26 or the method of any    one of items 24 to 26, wherein the compound of formula (I) is to be    administered in combination with a BRD4 inhibitor.

-   28. The compound for use according to item 27 or the pharmaceutical    composition for use according to item 27 or the use of item 27 or    the method of item 27, wherein the BRD4 inhibitor is CeMMEC2,    (S)-JQ1, I-BET 151, I-BET 762, PF-1, bromosporine, OTX-015, TEN-010,    CPI-203, CPI-0610, RVX-208, B12536, TG101348, LY294002, or a    pharmaceutically acceptable salt, solvate or prodrug of any one of    these agents.

-   29. The compound for use according to item 27 or the pharmaceutical    composition for use according to item 27 or the use of item 27 or    the method of item 27, wherein the BRD4 inhibitor is a compound    having the following structure:

-   -   or a pharmaceutically acceptable salt, solvate or prodrug        thereof.

-   30. In vitro use of a compound as defined in any one of items 1 to    20 as a TAF1 inhibitor.

-   31. A compound having any one of the following formulae:

-   -   or a pharmaceutically acceptable salt, solvate or prodrug        thereof.

-   32. A TAF1 inhibitor for use in therapy, wherein the TAF1 inhibitor    is to be administered in combination with a BRD4 inhibitor.

-   33. A BRD4 inhibitor for use in therapy, wherein the BRD4 inhibitor    is to be administered in combination with a TAF1 inhibitor.

-   34. A TAF1 inhibitor for use in the treatment or prevention of    cancer, wherein the TAF1 inhibitor is to be administered in    combination with a BRD4 inhibitor.

-   35. A BRD4 inhibitor for use in the treatment or prevention of    cancer, wherein the BRD4 inhibitor is to be administered in    combination with a TAF1 inhibitor.

-   36. A pharmaceutical composition comprising a TAF1 inhibitor and a    BRD4 inhibitor.

-   37. The pharmaceutical composition of item 36 for use in therapy.

-   38. The pharmaceutical composition of item 36 for use in the    treatment or prevention of cancer.

-   39. Use of a TAF1 inhibitor and a BRD4 inhibitor in the preparation    of a medicament for the treatment or prevention of cancer.

-   40. A method of treating or preventing cancer, the method comprising    administering a TAF1 inhibitor in combination with a BRD4 inhibitor    to a subject in need thereof.

-   41. The TAF1 inhibitor for use according to item 32 or 34 or the    BRD4 inhibitor for use according to item 33 or 35 or the    pharmaceutical composition of item 36 or the pharmaceutical    composition for use according to item 37 or 38 or the use of item 39    or the method of item 40, wherein the TAF1 inhibitor is a compound    as defined in any one of items 1 to 20.

-   42. The TAF1 inhibitor for use according to item 32, 34 or 41 or the    BRD4 inhibitor for use according to item 33, 35 or 41 or the    pharmaceutical composition of item 36 or 41 or the pharmaceutical    composition for use according to item 37, 38 or 41 or the use of    item 39 or 41 or the method of item 40 or 41, wherein the BRD4    inhibitor is CeMMEC2, (S)-JQ1, I-BET 151, I-BET 762, PF-1,    bromosporine, OTX-015, TEN-010, CPI-203, CPI-0610, RVX-208, 812536,    TG101348, LY294002, or a pharmaceutically acceptable salt, solvate    or prodrug of any one of these agents.

-   43. The TAF1 inhibitor for use according to item 34, 41 or 42 or the    BRD4 inhibitor for use according to item 35, 41 or 42 or the    pharmaceutical composition for use according to item 38, 41 or 42 or    the use of item 39, 41 or 42 or the method of any one of items 40 to    42, wherein said cancer is selected from prostate carcinoma, breast    cancer, acute myeloid leukemia, multiple myeloma, glioblastoma, and    NUT midline carcinoma.

The invention will now be described by reference to the followingexamples which are merely illustrative and are not to be construed as alimitation of the scope of the present invention.

EXAMPLES Example 1 Introduction

The compelling efficacy of compounds designed to target the twobromodomains of BRD4 in cancer models, such as the pan-BET inhibitorsJQ1 (Filippakopoulos et al., 2010) and I-BET-15 (Seal et al., 2012), hasprompted the development of drug candidates for these proteininteraction modules that are now undergoing clinical trials(Filippakopoulos et al., 2014). Despite the large number of competingclinical programs, the mechanistic and chemical diversity of currentlyavailable BRD4 inhibitors is limited (Filippakopoulos et al., Cell,2012; Filippakopoulos et al., 2014). Furthermore, there is a lack ofdetailed understanding of the factors affecting BRD4 function.

The inventors set out to design a strategy allowing the unbiasedscouting of high diversity chemical space for modulators of aBRD4-dependent inactive chromatin state. In the background of the humanhaploid cell line KBM7 (Andersson et al., 1995), allowing unambiguousmonoallelic genetic configurations, the RFP (Red Fluorescent Protein)gene was integrated in heterochromatic loci which are specificallyactivated by BRD4 inhibition. A high-diverse compound library of 89,355small molecules was then chosen and compounds were selected for theirability to reactivate RFP expression. The efficient identification ofmany BRD4 inhibitors, including all the BET inhibitors in this library,validated the experimental strategy. Importantly, the setup allowed theidentification of small molecules that efficiently induced RFPexpression but failed to bind BRD4, indicating a novel mechanism ofaction. As detailed further below, the inventors were able to show thatone such compound, CeMMEC1, functioned by binding the second bromodomainof the transcription initiation factor TAF1. Investigation of theproperties of this new compound and its derivatives enabled theinventors to demonstrate a strong synergy between the targeting of TAF1and BRD4, which resulted in efficient killing of BRD4-dependent cancercells.

Materials and Methods Cell Culture and Transfection

The human chronic myelogenous leukemia cell line KBM7 was cultured inIscove's Modified Dulbecco's Medium (IMDM, Gibco), supplemented with 10%Fetal Bovine Serum (FBS; Gibco) and 100 units/ml streptomycin andpenicillin (both from Gibco). The human embryonic kidney cell lineHEK293T was cultured in Dulbecco's Modified Eagles Medium (DMEM, Gibco)supplemented with 10% FBS and 100 units/ml streptomycin and penicillin.The peripheral blood human acute monocytic leukemia cell line THP1 andthe adenocarcinoma (non small lung cancer) cell lines H23 were culturedin RPMI-1640 (Roswell Park Memorial Institute, Gibco) supplemented with10% FBS and 100 units/ml streptomycin and penicillin. All the mentionedcell lines were incubated in 5% CO₂ atmosphere at 37° C.

HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen)according to the manufacturer's instructions.

Used plasmids were:

LZRS-RFP-ires-ZEO.

pFlag-CMV2-Brd4 (Addgene plasmid #22304).

Live Cell Imaging and Picture Quantification

Cells were seeded on clear flat bottom 96-well or 384-well plates(Corning) and treated with the indicated compounds for the specifiedconditions. Live cell imaging pictures were taken with the Operetta HighContent Screening System (PerkinElmer), 20× objective and non-confocalmode.

RFP quantification was done using the Harmony software (PerkinElmer) fornuclei detection and analysis, adapted for the nucleus diameter range ofthe specific cell line used (e.g. KBM7 nucleus diameter 13 μM). OnlyRFP-positive nuclei were detected and counted.

Apoptotic cells were detected using the Annexin V-FITC Apoptosisdetection kit (Abcam) according to the manufacturer's instruction.Apoptosis quantification was performed with the Harmony software(PerkinElmer) for nuclei and cytoplasm detection and analysis, adaptedfor the nucleus diameter range and cell shape of the specific cell lineused.

Western Blot

Proteins were separated on polyacrylamide gels with SDS running buffer(50 mM Tris, 380 mM Glycine, 7 mM SDS) and transferred to nitrocelluloseblotting membranes. All membranes were blocked with blocking buffer (5%(m/v) milk powder (BioRad) in TBST (Tris-Buffered Saline with Tween: 50mM Tris (tris (hydroxymethyl)aminomethane), 150 mM NaCl, 0.05% (v/v)Tween 20, adjusted to pH 7.6)). Proteins were probed with antibodiesagainst BRD4 (ab128874, 1:1000, Abcam), Actin (ab16039, 1:1000, Abcam),c-MYC (ab32072, 1:1000, Abcam), Flag (F1804, 1:1000, Sigma), BRD9(ab49313, 1:1000, Abcam) and Taf1 (sc-735, 1:1000, Santa Cruz), detectedby HRP (horseradish peroxidase) conjugated donkey anti-rabbit IgGantibody (ab16284, 1:5000, Abeam) or donkey anti-mouse IgG antibody(Pierce) and visualized with the Pierce ECL Western Blotting substrate(Amersham), according to the provided protocol.

RNA Extraction PCR and QPCR

RNA extraction was performed with TRIzol Reagent (Life Technologies)according to the manufacturer's protocol and Reverse Transcription (RT)was performed using the High Capacity cDNA Reverse Transcription Kit(Applied Biosystems) following the standard protocol.

Standard PCR was performed using Pfu DNA Polymerase (Fermenta) accordingto the standard conditions.

Standard PCR primers used:

WT-KBM7 genome (Sigma; forward 5′-CAGTTCCGCTACACGTGCTG,reverse 5′-CGTGGACCCTTAAAGAGAAGGT) REDS3 genome(Sigma; forward 5′-CAGTTCCGCTACACGTGCTG,reverse 5′-GCGCATGAACTCCTTGATGAC) Insulin_promoter(Sigma; forward 5′-CTCTCCTTGAGATGTTAATGTGGCT,reverse 5′-CACACGGAAGATGAGGTCCGAGTGG)

QPCR was performed using the Power SYBR Green Master mix (Invitrogen) asdescribed in the manufacturer's protocol.

QPCR primers used:

BRD4 (Sigma; forward 5′-CAGGAGGGTTGTACTTATAGCA,reverse 5′-CTACTGTGACATCATCAAGCAC). c-MYC(Sigma; forward 5′-GAAGGTGATCCAGACTCTGACCT,reverse 5′-CTTCTCTCCGTCCTCGGATTCT). Actin(Sigma; forward 5′-ATGATGATATCGCCGCGCTC, reverse 5′-CCACCATCACGCCCTGG).BRD3 (Sigma; forward 5′-AAGAAGAAGGACAAGGAGAAGG,reverse 5′-CTTCTTGGCAGGAGCCTTCT). TAF1(Sigma; forward 5′-TGCCCAGGAGATTGTGAACG, reverse 5′-GGCTTAGCCTGAGGCGTG).CREBP (Sigma; forward 5′-AGCAGCAGCTGGTTCTACTG,reverse 5′-CACAATGGGCAACTTGGCAG). EP300(Sigma; forward 5′-GCAGTGTGCCAAACCAGATG,reverse 5′-CATAGCCCATAGGCGGGTTG). STX2(Sigma; forward 5′-GGCAAGAAGGAAATTGATGTTCA,reverse 5′-AGACGTTCGGTTGTGCTTCT). RAN(Sigma; forward 5′-GAGAAGAACCACCTTGGGTGT,reverse 5′-TCCACCGAATTTCTCCTGGC). RFP(Sigma; forward 5′-GGGAGCGCGTGATGAACTTC,reverse 5′-GGAAGTTCACGCCGATGAAC).

Real-time amplification results were normalized to the endogenoushousekeeping genes Actin or GAPDH. The relative quantities werecalculated using the comparative CT (Cycle Threshold) Method (ΔΔCTMethod).

Cell Cycle Assay and Cell Sorting

For cell cycle analysis, cells were fixed with 70% ethanol for 24 hours,washed with PBS/0.1%-Tween and incubated with RNase for 20 minutes.Nuclei were stained with 5 μg/mI PI (propidium iodide, Sigma) for 10minutes prior to FACS analysis (BD FACSCalibur Flow Cytometer).

RFP-positive/negative cell sorting was performed using the FACSAria (BDBiosciences) sorter. Gates for positive or negative RFP populations weredone using the appropriate RFP-positive or negative controls.RFP-positive cells (1%, very positive population) were sorted inpresence of (S)-JQ1 0.5 μM 48 hours after infection. The negativepopulation was sorted in absence of (S)-JQ1 72 hours after the firstsorting (0.7%, negative, double-sorted single clones). RFP negativedouble-sorted clones were grown and treated with (S)-JQ1 0.5 μM severaltimes, in order to verify their ability to express RFP only upontreatment.

FISH Assay

The RFP specific probe (RFPprobe) was PCR performed using RFP specificprimers (Sigma; forward 5′-CGGTTAAAGGTGCCGTCTCG, reverse5′-AGGCTTCCCAGGTCACGATG) and labeled using dig-dUTP (DIG NickTranslation Mix, Roche).

Briefly, before hybridization, slides were fixed with 3%paraformaldehyde (Merck) in PBS for 10 min, permeabilized with 0.5%Triton (Sigma) in PBS for 5 min and then immediately passed through anethanol series (70%, 85% and 100%). The denaturation was performed in50% formamide (Sigma) in 2×SSC buffer (Saline Sodium Citrate buffer: 0.3M NaCl, 30 mM sodium citrate) simultaneously on nuclei and probes for 30min at 80° C. Hybridization was done overnight in a dark humiditychamber at 37° C. The slides were washed three times in 50%formamide/2×SSC buffer and another three times with 50% 2×SSC buffer(both at room temperature), incubated with Anti-Digoxigenin-Fluorescein(Fab fragments, Roche) for 1 hour at room temperature and detected withAlexaFluor488 IgG Fraction Monoclonal Mouse Anti-Fluorescein (JacksonLaboratory). Finally, nuclei were counterstained with4′,6-diamidino-2-phebyl-indole (DAPI, Sigma). Images were taken using aLeica DMI6000b inverted confocal system and a 63× 1.30 ACS Apo lens, andedited using Leica LAS AF software (Leica Microsystems) and Fiji(ImageJ).

Protein Expression of GST-Tagged BRD4

GST-BRD4 was extracted and purified from BL21 (DE3) E. coli cells (NewEngland BioLabs) and heat shock transformed with p5068 pGEX-6P-1 (fulllength BRD4 with GST-tag; Addgene). Transformed cells were inoculatedinto a LB agar plate with ampicillin 100 mg/ml. One colony was selectedand grown in LB broth (ampicillin 100 mg/rill) in a shaker (250 g) at37° C. until an OD (optical density) value of 0.8 at 600 nm was reached.Isopropyl-3-D-thiogalactopyranoside (IPTG; Sigma) was added to a finalconcentration of 0.3 mM, cultures were further grown for 3 hours at 37°C. Cells were harvested by centrifugation (6000 g for 15 min at 4° C.)and resuspended in cold lysis buffer (20 mM Tris-HCl pH 7.5, 0.5 M NaCl,5 mM EDTA, 1% Igepal) containing 2.5 mg/mI Lysozyme (Fluke), 0.1 mg/mlDNase I (Roche), 5 mM μ-mercaptoethanol and an appropriate amount ofprotease inhibitor cocktail (Roche). Cells were disrupted by gentlesonication (2 cycles, 10s) on ice and centrifuged (9000 g for 20 min at4° C.). BRD4 proteins carrying the GST-tag were purified under nativeconditions using Glutathione Sepharose 4B beads (GE Healthcare). TheGST-tagged proteins were eluted with elution buffer (10 mM Glutathione,50 mM Tris, pH 8.0, plus appropriate amount of protease inhibitorcocktail). The purity of the protein preparations was assessed bySDS-PAGE in 10% polyacrylamide gel, under reducing conditions.

AlphaLISA Assay

The Amplified Luminescent Proximity Homogenous Assay (AlphaLISA©) is thehomogenous and chemiluminescence-based method, used to explore thedirect interaction of the identified small molecules with BRD4 andtherefore, measure the IC₅₀ values of the direct BRD4 inhibitors.

Briefly, in this assay, the biotinylated histone peptide substrate iscaptured by streptavidin-coupled donor beads. The GST-tagged bromodomainis recognized and bound by an anti-GST antibody conjugated with anacceptor bead. In absence of an inhibitor, the bromodomain binds tohistone peptide substrate. The excitation (680 nm wavelength) of a donorbead provokes the release of a singlet oxygen molecule (¹O₂) thattriggers a cascade of energy transfer in the acceptor bead, resulting ina sharp peak of light emission at 615 nm. The event of a signal (alphacount) can only take place when the interaction partners are inproximity (<200 nm). The presence of a compound that blocks thehistone-docking site (inhibitor) results in the dropping of emission.

The AlphaLISA was performed for both bromodomains of BRD4 using the BRD4(BD1) Inhibitor Screening Kit (BPS Bioscience) and BRD4 (BD2) InhibitorScreening Kit (BPS Bioscience) following the manufacturer's protocol.For the GST full length BRD4 purified from BL21 cells, a mixture ofacetylated substrates from the BD1 and BD2 Inhibitor Screening Kitreported above was used.

Compounds were tested at a final assay concentration of 10 μM induplicates. To determine IC50 values, two-fold serial dilutions (12points; 50 μM to 0.02 μM) of test inhibitors were prepared. Reaction wasinitiated by adding one of the two bromodomains (BD1 or BD2) or the GSTfull length BRD4. After 30 minutes, GSH (Glutathione) Acceptor beads(PerkinElmer) were added and after another incubation time of 30minutes, Streptavidin-conjugated donor beads (PerkinElmer) were added.Alpha counts were read by EnVision 2104 Multilabel Reader (PerkinElmer).

Compound Screening

REDS3 cells were treated with the compound library (89,355 diversecompounds). The increase of RFP fluorescence, detected with the OperettaHigh Content Screening System (PerkinElmer), 20× objective andnon-confocal mode, was used as read-out.

Briefly, the screening was divided in three parts called respectively 1)primary screening, 2) follow up and 3) validation. During the primaryscreening REDS3 cells were treated with 10 μM of every compound, andlive cell imaging pictures were taken in order to assess their abilityto induce RFP expression 24 hours later. From this primary screening1,286 small molecules were selected as hits and re-screened in thefollow up part, in which REDS3 and WT-KBM7 were treated in 3-point doseresponse in order to exclude autofluorescent or toxic compounds. 80small molecules were selected as hits and used to treat WT-KBM7 andREDS3 in 8-point dose (2 fold dilution, starting from 100 μM) responseand 3-point time course (24/48/72 hours) in order to carefully selectthe best true hits (time- and dose-dependent RFP expression/noautofluorescence). UPLC-MS analysis was done to confirm purity and thecorrect mass of the small molecules selected; finally, 22 smallmolecules were chosen as screening hits.

Compound Synthesis

Compounds CeMMEC1 and CeMMEC2 were purchased from AKos GmbH (Steinen,Germany). Compound A1 was purchased from InterBioScreen Ltd.(Chernogolovka, Russia). Compounds A2, A3, A4 and A5 were purchased fromChemDiv (San Diego, USA). Synthesis of all other analogs was carried outby Enamine Ltd. (Kiev, Ukraine) following the scheme below:

A mixture of acid 1 (1.1 mmol), amine 2 (1.0 mmol), EDC (1.1 mmol), andHOBt (1.6 mmol) in DMF (1 ml) was stirred at room temperature for 24hours. Chloroform (6 ml) and water (8 ml) was added, organic layer wasseparated, washed with water (8 ml) twice, dried over Na₂SO₄ andevaporated. The crude residue was purified by reversed phase (C-18)chromatography with gradient elution (methanol-water) to yield pure 3.

All compounds were quality controlled by LC-MS, requiring a minimumpurity of 90%.

MS (m/z): [M+H]+ calcd. for C₁₉H₁₅ClN₂O₄ 371.0804, found 371.1.

Yield: 3%

¹H NMR (400 MHz, DMSO_d6) δ ppm 2.50 (br s, 12H) 3.39 (br s, 5H) 3.59(s, 3H) 3.66 (s, 1H) 6.01 (s, 2H) 6.91 (br d, J=8.39 Hz, 1H) 7.13 (br d,J=8.39 Hz, 1H) 7.43 (s, 1H) 7.57 (br t, J=7.46 Hz, 1H) 7.76 (br t,J=7.46 Hz, 1H) 8.06 (s, 1H) 8.15 (br d, J=7.93 Hz, 1H) 8.29 (br d,J=7.93 Hz, 1H) 10.26 (br s, 1H); MS (m/z): [M+H]+ calcd. for C₁₈H₁₄N₂O₄323.1026, found 323.2.

Yield: 10%

MS (m/z): [M+H]+ calcd. for C₂₀H₁₇ClN₂O₄ 385.094961, found 385.0.

Yield: 3%

¹H NMR (400 MHz, DMSO_d6) δ ppm 2.10 (br s, 2H) 2.50 (br s, 24H) 3.33(s, 9H) 3.59 (s, 3H) 4.10 (dt, J=17.60, 4.72 Hz, 4H) 6.93-7.00 (m, 1H)7.23-7.30 (m, 1H) 7.44 (br s, 1H) 7.52-7.61 (m, 1H) 7.76 (br t, J=7.46Hz, 1H) 8.05 (s, 1H) 8.13 (br d, J=7.93 Hz, 1H) 8.29 (br d, J=7.93 Hz,1H) 10.25 (s, 1H); MS (m/z): [M+H]+ calcd. for C₂₀H₁₈N₂O₄ 351.1339,found 351.2.

Yield: 39%

¹H NMR (400 MHz, DMSO_d6) δ ppm 2.50 (br s, 13H) 3.33 (br s, 7H) 3.59(s, 3H) 3.75 (s, 3H) 6.94 (br d, J=8.39 Hz, 2H) 7.57 (br t, J=7.46 Hz,1H) 7.64 (br d, J=8.39 Hz, 2H) 7.76 (br t, J=7.69 Hz, 1H) 8.06 (s, 1H)8.16 (br d, J=7.93 Hz, 1H) 8.29 (br d, J=7.93 Hz, 1H) 10.21 (s, 1H); MS(m/z): [M+1-1]+ calcd. for C₁₈H₁₆N₂O₃ 309.1234, found 309.2.

Yield: 20%

¹H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 2.49 (br s, 4H) 3.01 (s, 6H) 3.62(s, 3H) 7.21 (br d, J=8.53 Hz, 2H) 7.50 (br t, J=7.28 Hz, 1H) 7.69 (brt, J=7.03 Hz, 1H) 7.85 (br d, J=9.03 Hz, 2H) 7.98 (s, 1H) 8.17-8.36 (m,2H) 10.28 (s, 1H); MS (m/z): [M+H]+ calcd. for C₁₈H₁₃F₃N₂O₃ 363.0951,found 363.2.

Yield: 8%

¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.53-2.55 (m, 1H) 3.28-3.34 (m, 2H) 3.60(s, 3H) 3.76 (s, 3H) 6.69 (br d, J=6.86 Hz, 1H) 7.23-7.34 (m, 2H) 7.43(br s, 1H) 7.57 (br t, J=7.55 Hz, 1H) 7.77 (br t, J=7.55 Hz, 1H) 8.08(s, 1H) 8.16 (br d, J=8.23 Hz, 1H) 8.30 (br d, J=8.23 Hz, 1H) 10.31 (brs, 1H); MS (m/z): [M+H]+ calcd. for C₁₈H₁₆N₂O₃ 309.1234, found 309.2.

Yield: 29%

¹H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 1.21 (s, 1H) 2.46 (br s, 3H) 2.99(s, 5H) 3.59 (s, 3H) 7.16-7.35 (m, 1H) 7.47 (br t, J=7.53 Hz, 1H) 7.66(br t, J=7.65 Hz, 1H) 8.01 (s, 1H) 8.15-8.30 (m, 4H) 8.76 (br s, 1H)10.27 (br s, 1H); MS (m/z): [M+H]+ calcd. for C₁₆H₁₃N₃O₂ 280.1081, found280.2.

Yield: 4%

¹H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 1.25 (br s, 1H) 1.32 (br d, J=8.86Hz, 1H) 2.41-2.58 (m, 4H) 3.03 (br s, 7H) 3.63 (s, 3H) 3.78 (s, 6H) 6.17(br s, 1H) 7.00 (br d, J=1.87 Hz, 2H) 7.51 (br t, J=7.46 Hz, 1H) 7.70(br t, J=7.23 Hz, 1H) 7.97 (s, 1H) 8.20-8.37 (m, 2H) 10.02 (s, 1H); MS(m/z): [M+H]+ calcd. for C₁₉H₁₈N₂O₄ 339.1339, found 339.2.

Yield: 37%

¹H NMR (500 MHz, DMSO-d₆) δ ppm 2.34-2.66 (m, 5H) 3.30-3.34 (m, 1H) 3.59(s, 3H) 5.36 (s, 2H) 7.20 (br s, 1H) 7.57 (br t, J=7.41 Hz, 1H) 7.81 (brt, J=7.68 Hz, 1H) 8.27 (br d, J=7.68 Hz, 1H) 8.47 (s, 1H) 8.69 (br d,J=8.51 Hz, 1H); MS (m/z): [M+H]+ calcd. for C₁₄H₁₂N₄O₄ 301.0931, found301.2.

Yield: 10%

¹H NMR (500 MHz, DMSO-d₆) δ ppm 0.84-0.93 (m, 2H) 1.06 (br dd, J=8.23,2.47 Hz, 2H) 2.08 (br s, 1H) 2.48-2.52 (m, 8H) 3.33 (s, 4H) 3.57 (s, 3H)7.54-7.63 (m, 1H) 7.79 (br t, J=7.55 Hz, 1H) 8.25-8.35 (m, 3H)12.21-12.37 (m, 1H); MS (m/z): [M+H]+ calcd. for C₁₆H₁₄N₄O₃ 311.1139,found 311.0.

Yield: 8%

¹H NMR (400 MHz, CDCl₃) δ ppm 1.23 (br s, 1H) 1.67-2.15 (m, 4H) 3.29 (brs, 3H) 3.82 (br s, 4H) 3.90 (br d, J=6.78 Hz, 1H) 3.96-4.04 (m, 2H) 4.32(br s, 1H) 6.84 (br d, J=8.03 Hz, 1H) 7.16 (br d, J=8.03 Hz, 1H) 7.35(br s, 1H) 7.40-7.48 (m, 1H) 7.51 (br s, 1H) 7.62 (br t, J=6.50 Hz, 1H)8.02 (br d, J=7.78 Hz, 1H) 8.30 (br d, J=7.28 Hz, 1H) 8.78 (br s, 1H);MS (m/z): [M+H]+ calcd. for C₂₃H₂₄N₂O₆ 409.1758, found 409.2.

Yield: 5%

¹H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 1.25 (br s, 1H) 2.30 (s, 6H) 2.51(br s, 3H) 2.71 (br t, J=6.10 Hz, 2H) 3.02 (br s, 3H) 3.63 (s, 3H) 3.80(s, 3H) 4.05 (br t, J=6.24 Hz, 2H) 6.84 (br d, J=8.86 Hz, 1H) 7.21 (brd, J=8.71 Hz, 1H) 7.42-7.55 (m, 2H) 7.69 (br t, J=7.95 Hz, 1H) 7.95 (s,1H) 8.29 (br dd, J=12.59, 8.39 Hz, 2H) 9.93 (br s, 1H); MS (m/z): [M+H]+calcd. for C₂₂H₂₅N₃O₄ 396.1918, found 396.2.

Yield: 16%

¹H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 1.05 (br t, J=7.00 Hz, 6H) 2.61 (q,J=7.00 Hz, 3H) 2.55-2.69 (m, 1H) 2.85 (br t, J=6.30 Hz, 2H) 3.02 (br s,5H) 3.63 (s, 3H) 3.79 (s, 3H) 4.00 (br t, J=6.53 Hz, 2H) 6.83 (br d,J=8.86 Hz, 1H) 7.20 (br d, J=8.86 Hz, 1H) 7.42-7.59 (m, 2H) 7.69 (br t,J=7.46 Hz, 1H) 7.94 (s, 1H) 8.29 (br dd, J=12.36, 8.63 Hz, 2H) 9.92 (s,1H); MS (m/z): [M+H]+ calcd. for C₂₄H₂₉N₃O₄ 424.2231, found 424.2.

Yield: 5%

¹H NMR (400 MHz, Solvent) 6 ppm 1.92 (br t, J=6.06 Hz, 2H) 2.50 (br s,11H) 2.75 (br t, J=6.06 Hz, 2H) 3.33 (br s, 6H) 3.59 (s, 3H) 4.06-4.16(m, 2H) 6.72 (br d, J=8.86 Hz, 1H) 7.35 (br d, J=8.86 Hz, 1H) 7.48 (brs, 1H) 7.56 (br t, J=7.46 Hz, 1H) 7.76 (br t, J=7.46 Hz, 1H) 8.03 (s,1H) 8.15 (br d, J=7.93 Hz, 1H) 8.29 (br d, J=7.93 Hz, 1H) 10.11 (s, 1H);MS (m/z): [M+H]+ calcd. for C₂₀H₁₈N₂O₃ 335.1390, found 335.1.

Yield: 16%

¹H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 2.41-2.61 (m, 2H) 3.04 (br s, 2H)3.70 (s, 3H) 4.25 (br d, J=3.26 Hz, 4H) 6.64-6.84 (m, 2H) 7.16 (br d,J=8.39 Hz, 1H) 7.26 (br t, J=7.23 Hz, 1H) 7.37 (br s, 1H) 7.52 (br d,J=8.39 Hz, 1H) 7.57-7.75 (m, 1H) 7.90 (br d, J=7.46 Hz, 1H) 10.38 (br s,1H) 12.76-12.79 (m, 1H); MS (m/z): [M+H]+ calcd. for C₁₉H₁₆N₂O₄337.1183, found 337.2.

Yield: 95%

¹H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 2.41-2.58 (m, 2H) 2.96-3.05 (m, 3H)3.08 (br s, 1H) 3.55 (s, 3H) 4.27 (br s, 1H) 4.33 (br d, J=2.33 Hz, 4H)6.54 (br d, J=7.93 Hz, 1H) 6.91 (d, J=8.39 Hz, 1H) 7.32 (br d, J=7.46Hz, 1H) 7.47 (br t, J=7.93 Hz, 1H) 7.52-7.63 (m, 2H) 7.70 (br d, J=7.46Hz, 1H) 8.09-8.26 (m, 1H) 9.96 (s, 1H); MS (m/z): [M+H]+ calcd. forC₁₉H₁₆N₂O₄ 337.1183, found 337.2.

Yield: 87%

MS (m/z): [M+H]+ calcd. for C₁₅H₁₅N₃O₅ 318.108447, found 318.2.

Yield: 90%

¹H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 2.51 (br s, 1H) 3.04 (s, 2H) 3.54(s, 3H) 4.23 (br d, J=4.20 Hz, 4H) 6.38 (br d, J=9.79 Hz, 1H) 6.72 (brd, J=8.86 Hz, 1H) 7.07 (br dd, J=8.86, 2.33 Hz, 1H) 7.26 (br d, J=2.33Hz, 1H) 7.95 (br dd, J=9.33, 2.33 Hz, 1H) 8.45 (br d, J=2.33 Hz, 1H)9.58 (s, 1H); MS (m/z): [M+H]+ calcd. for C₁₅H₁₄N₂O₄ 287.1026, found287.1.

Yield: 22%

MS (m/z): [M+H]+ calcd. for C₁₄H₁₃N₃O₅ 304.092797, found 304.0.

Yield: 88%

¹H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 2.51 (br s, 2H) 3.02 (br s, 3H)3.28 (s, 3H) 4.23 (br d, J=4.20 Hz, 4H) 6.73 (br d, J=8.39 Hz, 1H) 6.92(br dd, J=8.86, 2.33 Hz, 1H) 7.30 (br d, J=2.33 Hz, 1H) 8.26 (s, 1H)10.75 (s, 1H); MS (m/z): [M+H]+ calcd. for C₁₄H₁₃N₃O₅ 304.0928, found304.0.

Yield: 21%

¹H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 1.20 (br t, J=7.46 Hz, 3H)2.42-2.59 (m, 4H) 2.79 (s, 1H) 2.95 (s, 1H) 3.03 (br s, 3H) 3.50 (s, 1H)3.55 (s, 2H) 4.24 (br d, J=4.66 Hz, 4H) 6.72 (br d, J=8.86 Hz, 1H) 7.07(br dd, J=8.86, 2.33 Hz, 1H) 7.26 (br d, J=2.33 Hz, 1H) 7.77 (br s, 1H)7.91 (s, 1H) 8.32 (br d, J=2.33 Hz, 1H) 9.54 (s, 1H); MS (m/z): [M+H]+calcd. for C₁₇H₁₈N₂O₄ 315.1339, found 315.1.

Yield: 87%

1H NMR (400 MHz, DMSO_d6+CCl₄) δ ppm 1.71 (br s, 4H) 2.43 (br s, 2H)2.47-2.56 (m, 2H) 2.72 (br s, 2H) 3.03 (s, 2H) 3.47 (s, 3H) 4.23 (br d,J=4.20 Hz, 4H) 6.70 (br d, J=8.39 Hz, 1H) 7.05 (br d, J=8.86 Hz, 1H)7.26 (s, 1H) 7.77 (s, 1H) 9.71 (s, 1H); MS (m/z): [M+H]+ calcd. forC₁₉H₂₀N₂O₄ 341.1496, found 341.1.

Yield: 91%

¹H NMR (400 MHz, Solvent) δ ppm 1.25 (br s, 4H) 2.51 (br s, 16H) 3.01(br s, 27H) 3.63 (s, 3H) 3.81 (br d, J=13.99 Hz, 6H) 6.83 (br d, J=7.93Hz, 1H) 7.19 (br s, 1H) 7.44-7.55 (m, 2H) 7.69 (t, J=7.50 Hz, 1H) 7.95(s, 1H) 8.22-8.34 (m, 2H) 9.95 (br s, 1H); MS (m/z): [M+H]+ calcd. forC₁₉H₁₈N₂O₄ 339.1339, found 339.2.

Yield: 81%

RNA-Sequencing

The amount of total RNA was quantified using Qubit 2.0 FluorometricQuantitation system (Life Technologies) and the RNA integrity number(RIN) was determined using Experion Automated Electrophoresis System(Bio-Rad). RNA-seq libraries were prepared with TruSeq Stranded mRNA LTsample preparation kit (Illumina) using Sciclone and Zephyr liquidhandling robotics (PerkinElmer). Library amount was quantified usingQubit 2.0 Fluorometric Quantitation system (Life Technologies) and thesize distribution was assessed using Experion Automated ElectrophoresisSystem (Bio-Rad). For sequencing libraries were pooled and sequenced onIllumina HiSeq 2000 using 50 bp single-read. Reads were aligned withtophat (v2.0.4) with the —no-novel-juncs —no-novel-indels options (Kimet al., 2013). Gene expression was calculated as Reads Per Kb perMillions of reads (RPKMs) using RPKM_count.py from RSeQC package (WangL. et al., 2012) and the NCBI RNA reference sequences collection(RefSeq) downloaded from UCSC (Kent et al., 2002). The enrichmentcalculation was done by Gene Set Enrichment Analysis (Subramanian etal., 2005; Mootha et al., 2003).

TAF1 Binding Assay

TAF1 binding assays were conducted using the EPIgeneous™ Binding Domainkit B (Cisbio Bioassays) according to manufacturer's instructions.Binding was determined by the displacement of an acetylatedbiotin-peptide from a GST tagged TAF1 protein using HTRF with a Eu3+conjugated GST antibody donor and streptavidin conjugated acceptor.Compounds were dispensed into assay plates, ProxiPlate-384 Plus (PerkinElmer) using an Echo 525 Liquid Handler (Labcyte). Binding assays wereconducted in a final volume of 20 μl with 5 nM TAF1-GST, 50 nM peptide(SGRGK (ac)GGK (ac)GLGK (ac)GGAK (ac)RHRK (biotin)-acid), 6.25 nMStreptavidin-XL665, 1:200 Anti-GST-Eu3+ cryptate and 0.1% DMSO. Assayreagents were dispensed into plates using a Multidrop combi (ThermoScientific) and incubated at room temperature for 3 hours. Fluorescencewas measured using a PHERAstar microplate reader (BMG) using the HTRFmodule with dual emission protocol (A=ex. 320 nm em. 665 nm, B=ex. 320nm em. 620 nm). Raw data were processed to give an HTRF ratio (channelA/B*10000), which was used to generate IC50 curves.

Protein Expression and Purification of TAF1 Second Bromodomain

TAF1 second bromodomain (Uniprot P21675, residues 1501-1634) was clonedinto a pET28 derived expression vector, pNIC28-Bsa4 using ligationindependent cloning. Colonies transformed in competent E. coli BL21(DE3)-R3-pRARE2 cells (phage-resistant derivative of BL21 (DE3) strain),with a pRARE plasmid encoding rare codon tRNAs were grown overnight at37° C. in 10 ml of Terrific broth medium (Sigma) with 50 μg/ml kanamycinand 34 μg/ml chloramphenicol. Cells were grown at 37° C. in TB fromovernight cultures until A600 reached between 0.8-1.1, then the mediawas cooled and 0.2 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) wasadded to induce the protein expression at 18° C. for 16 hours. Thebacteria were harvested by centrifugation (JLA 8,100 rotor BeckmanCoulter Avanti J-20 XP centrifuge) and were frozen at −20° C. Cellexpressing 6×His tagged TAF1 second bromodomain were re-suspended inlysis buffer (20 mM Hepes pH 7.5, 500 mM NaCl, 10 mM Imidazole, 5%glycerol and 0.2 mM TCEP (Tris (2-carboxyethyl)phosphine hydrochloride)in the presence of protease inhibitors cocktail (1 μl/ml) and lysedusing an EmulsiFlex-05 high pressure homogenizer (Avestin-Mannheim,Germany) at 4° C. The lysate was cleared by centrifugation (14,000×g for1 hour at 4° C.). After centrifugation, the supernatant was loaded ontothe nickel column and eluted in an imidazole linear gradient. The elutedprotein was collected and treated overnight with TEV protease at 4° C.to remove the N terminal tag. Digested protein was loaded onto a nickelcolumn again to remove the non-cleaved protein and the hexa-histidineTEV used. The flow through containing the untagged protein was collectedand further purified through a size exclusion chromatography in 20 mMHepes pH 7.5, 500 mM NaCl, 5% glycerol and 0.2 mM TCEP (HiLoad 16/60Superdex 75 GE Healthcare Life Sciences). Similarly, GST-tagged TAF1second bromodomain was purified using a 5 ml Glutathione Sepharose FastFlow column with elution buffer of 50 mM Tris pH8, 10 mM reducedglutathione. Gel filtration (HiLoad 16/60 Superdex 200) chromatographywas performed as the final purification step. The correct mass andpurity for both constructs were confirmed by an Agilent 1100 SeriesLC/MSD TOF (Agilent Technologies Inc. Palo Alto, Calif.).

Isothermal Titration calorimetry

calorimetric experiments were performed on a VP-ITC micro-calorimeter(MicroCal™, LLC Northampton, Mass.). TAF1 (2) was buffer exchanged bydialysis into buffer 20 mM Hepes pH 7.5, 150 mM NaCl, and 0.5 mM TCEP.All measurements were carried out at 293.15 K while stirring at 286 rpm.The micro syringe was loaded with a protein solution of 295 μM, thecompound solution was prepared at 25 μM and 2 ml for the cell. Allinjections were performed using an initial injection of 2 μl followed by34 injections of 8 μl with a duration of 16 seconds per injection and aspacing of 240 seconds between injection. The data were analysed withthe MicroCal ORIGIN software package employing a single binding sitemodel. The first data point was excluded from the analysis.Thermodynamic parameters were calculated (ΔG=ΔH−TΔS=−RTlnK_(B) where ΔG,ΔH and ΔS are the changes in free energy, enthalpy and entropy ofbinding, respectively).

Molecular Modeling

The crystal structures of the second bromodomain of TAF1, of ATAD2 andof BRD4 were downloaded from the RCSB protein data bank (pdb:4qst,pdb:3uv4 and pdb:3mxf). The structures were corrected, protonated andenergy minimized using the LigX workflow of the molecular modelingsoftware MOE (Molecular Operating Environment (MOE), 2014; ChemicalComputing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC,Canada, H3A 2R7). The hit compounds were prepared with the washing toolin MOE.

For binding pose prediction, the template-based docking protocol of MOEwas used. CeMMEC1 was docked into the crystal structure of the secondbromodomain of TAF1 (pdb:3uv4) (Filippakopoulos et al., Cell, 2012)using the atom positions of 1-methylquinolin 2-one bound to thebromodomain of ATAD2 (pdb:4qst) (Chaikuad et al., 2014) as template forpose prediction (Picaud et al., 2015). Similarly, CeMMEC2 was dockedinto a crystal structure of JQ1 bound to BRD4 (pdb:3mxf)(Filippakopoulos et al., 2010), with the triazole ring serving as poseprediction template.

Results A Cellular Reporter for Detection of Functional BRD4 Inhibition

The inventors aimed to generate a cellular reporter system that rapidlyresponds to epigenetic changes with a gain of signal and is optimallysuited to both chemical and genetic screens. Therefore, they developed astrategy of targeting a reporter construct to heterochromatic loci inKBM7 cells, a chronic myeloid leukaemia cell line with a near-haploidkaryotype (Andersson et al., 1995) (see FIG. 1a ). To identify suchBRD4-repressed loci in an unbiased way, KBM7 cells were pre-incubatedwith the potent and selective BET bromodomain inhibitor (S)-JQ1 at aconcentration that was sufficient to provoke chromatin reorganization,c-MYC repression (see FIG. 6a ) and partial cell cycle arrest, while notcausing apoptosis (see FIG. 6b ). (S)-JQ1-treated cells were theninfected with a retrovirus for the expression of RFP and a strategy ofdouble FACS sorting was applied to obtain a population of cells thatexpress RFP in the presence of (S)-JQ1 and repress the transgene afterwithdrawal of the compound (see FIGS. 1a and 1b ).

Three clones were isolated as Reporters for Epigenetic Drug Screening(REDS1, REDS2 and REDS3), which expressed RFP in response to (S)-JQ1(see FIG. 6c ). Because of its strong and uniform RFP intensity, cloneREDS3 was selected for further validation and experiments. The treatmentof REDS3 cells with (S)-JQ1 induced a clear and robust increase of RFPexpression detected by flow cytometry (see FIG. 1c ), live cell imaging(see FIG. 6c ) and real time PCR (RT-PCR) (see FIG. 6d ). In addition toRFP, the zeocin resistance gene present on the retroviral vector wasalso upregulated (RT-PCR data, FIG. 6e ). Moreover, RFP expression wasnot caused by the partial cell cycle arrest induced by (S)-JQ1, as thesynchronization of REDS3 in different phases of the cell cycle did notincrease the number of RFP-positive cells (see FIG. 6f ). Recently, theactivation of the LTR (Long Terminal Repeat) of HIV-1 (HumanImmunodeficiency Virus-1) has been reported to be stimulated by (S)-JQ1,and the inhibition of BRD4 potentiates the action of knowntranscriptional HIV-1 reactivating compounds, such as PMA (phorbolmyristate acetate) or PHA (phytohemagglutinin) (Zhu et al., 2012;Banerjee et al., 2012). To rule this out as a possible mechanism of RFPexpression, REDS3 cells were treated with PMA, PHA or a combination ofeach of them with (S)-JQ1. No increase of RFP-positive nuclei wasobserved with these compounds (see FIG. 6g ). Therefore, RFP expressionwas likely due to the effect of (S)-JQ1 on the locus of insertion ratherthan the inserted LTR.

(S)-JQ1, like almost all BRD4 inhibitors, targets the entire BETbromodomain family (BRD2, BRD3, BRD4, BRDT) with comparable potency inaddition to very weak interaction with a few other human bromodomains(Filippakopoulos et al., 2010). To clarify which BET target isresponsible for RFP repression and rule out off-target effects, all the(S)-JQ1 targets were knocked down individually in the REDS3 clone andRFP-positive cells were quantified by flow cytometry. Only thedownregulation of BRD4 resulted in an increase of RFP-positive nuclei(see FIG. 1d ). This effect was also visible by live cell imaging (seeFIG. 1e ) and accompanied by increased levels of RFP mRNA, not observedfor instance following BRD3 downregulation (see FIG. 6h ). Thus, it hasbeen possible to create an experimental system allowing for a focusedphenotypic screen for perturbations, be they genetic, pharmacological ormetabolic, resulting in release from the BRD4-driven heterochromatinstate.

BRD4 Inhibition Upregulates Genes Flanking Super-Enhancer Regions

To further validate the REDS3 clone, FISH (fluorescence in situhybridization) was performed and the presence of a single RFP insertionper cell was confirmed (see FIG. 2a ). The RFP probe was preferentiallylocated in proximity to the nuclear membrane (see FIG. 2b ), indicatingRFP heterochromatin localization (Schermelleh et al., 2008; Towbin etal., 2012). A sequencing approach was used to map the RFP locus toregion 12q24.33, located less than 3 Mb from the telomere of chromosome12 (see FIG. 2c ). The sequencing data were confirmed by PCR usingspecific pairs of genomic primers for wild-type (WT) and REDS3 KBM7cells (see FIG. 2d ). Less than 5 kb upstream of the RFP insertion isthe STX2 gene, which is lowly expressed in KBM7 (RNA-seq (RNAsequencing) data; RPKM<1) and flanked by heterochromatin regions (Nature2012, 489, 57-74). In contrast, the gene RAN, located 35 kb downstreamof the RFP locus, is robustly expressed in KBM7 and has previously beendescribed as a BRD4 target gene (Nagarajan et al., 2014). Interestingly,the region between STX2 and RAN is enriched in repeated enhancersequences (ENCODE), endorsing the hypothesis of a super-enhancer (Pottet al., 2015; Whyte et al., 2013) controlling the expression of RAN.Even though BRD4 has been considered a transcriptional activator, REDScells respond to BRD4 inhibition with activation of RFP. It wastherefore asked whether BRD4 inhibitors directly upregulated othergenes. According to RNA-seq of KBM7 cells treated for 24 hours with 1 μM(S)-JQ1, 133 genes were significantly upregulated more than two-fold(see FIG. 2e ). Other cell lines (MOLM-13, KASUMI-1, MV4-11, MOLT-3,MEG-01, K-562) responded similarly, and e.g. in MOLM-13 cells 172 geneswere upregulated after only 2 hours with (S)-JQ1. Remarkably, functionalannotation (Huang et al., Nat. Protoc., 2009, 4, 44-57; Huang et al.,Nucleic Acids Res., 2009, 37, 1-13) of the (S)-JQ1 upregulated gene setrevealed a strong cell line-independent enrichment of genes involved inchromatin remodelling (see FIG. 2f ), particularly histone genes,corroborating the hypothesis of a global chromatin reorganizationfollowing BRD4 inhibition.

The expression of the genes proximal to the RFP insertion site was thenchecked in WT-KBM7 cells treated with (S)-JQ1, in order to see if achromatin remodelling process was occurring at this locus wheninhibiting BRD4. In line with the inventors' hypothesis, RAN expressiondecreased while STX2 mRNA levels increased upon (S)-JQ1 treatment (seeFIG. 2g ), indicating that BRD4 inhibition not only results in thereduced expression of RAN, but also raises the transcription of STX2.

Screening for Functional BRD4 Inhibitors

Although BRD4 is well studied, currently available inhibitors are oflimited structural and mechanistic diversity (Filippakopoulos et al.,2014; Filippakopoulos et al., Bioorganic Med. Chem., 2012), anddruggable targets upstream or downstream of BRD4 have remained elusive.The inventors aimed to screen for small molecules able to functionallyinhibit BRD4. In order to confirm the specificity of the reporterdetecting BRD4 inhibition, and not any other epigenetic pertubations,REDS3 was treated with several chromatin-targeted molecules. Within thissmall panel of compounds, only BET inhibitors were able to activate RFPexpression (see FIG. 3a ). With the high specificity of this reportercell line confirmed, a large live cell imaging screen was performed,testing 89,355 small molecules (see FIGS. 7a and 10) for their abilityto induce the expression of RFP in REDS3 cells after 24 hours. 0.5 μM(S)-JQ1 was used as positive control, as this was the lowestconcentration causing full activation of RFP signal (see FIG. 7b ) andan excellent Z′-factor (Running et al., 1999) (see FIGS. 7c and 10).Following hit validation and elimination of autofluorescent compounds,22 compounds were confirmed as screening hits (see FIGS. 3b and 11).Remarkably, all BRD4 inhibitors contained in the compound library((S)-JQ1 (Filippakopoulos et al., 2010), PFI1 (Fish et al., 2012),I-BET151 (Seal et al., 2012), I-BET-762 (Mirguet et al., 2013),Bromosporine, OXT015, RVX208 (McLure et al., 2013), BI-2536 (Ciceri etal., 2014) and TG-101348 (Ciceri et al., 2014)) were part of this group,underscoring the validity of the setup. 13 compounds were new, amongwhich the inventors suspected new BRD4-inhibition scaffolds or evenagents with new mechanism of action. RT-PCR for c-MYC revealed that twoout of those small molecules were capable of reducing the expression ofthis oncogene in a dose-dependent manner to a level comparable to(S)-JQ1 treatment (see FIGS. 3c and 11). These two compounds werestructurally distinct from (S)-JQ1 and all the other BRD4 inhibitors.The inventors named them CeMM Epigenetic Compounds CeMMEC1 and CeMMEC2(see FIG. 3d ). REDS3 cells treated with CeMMEC1 and CeMMEC2 expressedRFP detected by live cell imaging in a dose-dependent manner (see FIGS.7d and 7e ). Transcriptome-wide effects of CeMMEC1 and CeMMEC2 weremeasured and compared to (S)-JQ1. While the number of transcriptsregulated by CeMMEC1 and CeMMEC2 is lower than for (S)-JQ1, there is asignificant overlap of the altered gene sets (see FIG. 7f ) and a goodcorrelation between the regulated genes (see FIG. 7g ). Overall thesedata indicate that these two compounds belong to new chemical structuralclasses of functional BRD4 inhibitors. BRD4 inhibitors are mainlydeveloped for applications in oncology, where they reduce theproliferation of certain cancer cells. Therefore, the inventors treatedTHP1 cells, a human acute monocytic leukemia cell line sensitive to theinhibition of BRD4, with (S)-JQ1, CeMMEC1 and CeMMEC2 and analyzed cellcycle profiles and induction of apoptosis after 48 and 72 hoursrespectively. The cell cycle assay showed a clear and dose-dependentdecrease of the number of cells in S-phase, indicative of G1-phase cellcycle arrest with all three compounds (see FIG. 3e ). Moreover, allcompounds induced apoptosis, as judged by AnnexinV staining (see FIG. 3f). In terms of potency, (S)-JQ1 showed the strongest effects, followedby CeMMEC2 and CeMMEC1.

Molecular Characterization of Functional BRD4 Inhibitors

To investigate whether CeMMEC1 and CeMMEC2 inhibit BRD4 through directphysical engagement, their ability to compete for binding of the BRD4bromodomains to an acetylated histone peptide was tested in an AmplifiedLuminescent Proximity Homogenous Assay (AlphaLISA) immunoassay(Bielefeld-Sevigny, 2009). CeMMEC1 was neither able to bind the firstnor the second bromodomain of BRD4, as no decrease of fluorescence wasobserved when this compound was added to the assays (see FIG. 4a ). Incontrast, CeMMEC2 bound both bromodomains of BRD4, comparably to (S)-JQ1(see FIG. 4a ), when used at 10 μM. Dose response AlphaLISA assaysperformed with full length BRD4 (GST-BRD4) showed that CeMMEC2 has anIC50 of 0.9 μM compared to 0.2 μM of (S)-JQ1 (see FIG. 4b ). Similarresults were obtained when the individual bromodomains of BRD4 weretested separately (see FIG. 8a ).

To comprehensively analyze the binding capability of CeMMEC1 and CeMMEC2to representative bromodomain proteins, BromoScan profiles were obtained(see FIG. 4c ). Similarly to other BRD4 inhibitors, CeMMEC2 not onlybound BRD4 but also all other proteins of the BET family. In contrast,CeMMEC1 only bound BRD4 very weakly, in line with the AlphaLISA data.Surprisingly, this compound showed high affinity for the bromodomains ofCREBBP, EP300, BRD9, and the second bromodomain of TAF1 (TAF1 (2)), alsoconfirmed by the sub-micromolar binding constants (see FIG. 8b ).Recently CREBBP and EP300 have been described as BRD4 cofactors inregulation of transcriptional control (Roe et al., 2015), while theinterplay between BRD4 and BRD9 or TAF1 has not been reported yet. Sincethese four bromodomain containing proteins are direct targets ofCeMMEC1, it was asked whether the loss of one of them could mimic BRD4inhibition and increase RFP expression in REDS3 cells. CREBBP, EP300,BRD9 and TAF1 were knocked down in REDS3 cells, using two independentshRNA hairpins for each gene. Western Blot was performed to confirm thelevel of downregulation by each hairpin (see FIG. 4d ). The number ofRFP-positive nuclei after knockdown was quantified by live cell imaging.A significant increase of RFP-positive nuclei was observed whendownregulating TAF1, whereas no increase of RFP-positive nuclei wasdetected with BRD9, CREBBP or EP300 downregulation (see FIG. 4e ).Moreover, as chemical probes for CREBBP and EP300 have already beenreported (Hay et al., 2014; Hammitzsch et al., 2015; Picaud et al.,2015), the inventors tested them in dose response to see if a furtherdecrease of the activity of these bromodomains could raise the number ofRFP-positive cells (see FIG. 4f ). No RFP expression was detected usingI-CBP112, the most selective CREBBP/EP300 inhibitor. CBP30 is known tobind BRD4 at high concentrations. Accordingly, doses able to inhibitCREBBP and EP300 (Hammitzsch et al., 2015) did not show any effect,while treatment with 10 μM CBP30 resulted in a 1.5 fold increase ofRFP-positive cells compared to DMSO treated cells, likely due to BRD4inhibition.

Several BRD4 inhibitors, including bromosporine and a3,5-dimethylisoxazole derivative (McKeown et al., 2014), are known tobind TAF1, but currently, there is no specific inhibitor available forthis bromodomain containing protein. Given that CeMMEC1 showed highaffinity for TAF1 (2), the inventors decided to further characterizethis interaction. Using the BromoKdELECT assay, it was confirmed thatCeMMEC1 binds to TAF1 (2), with a Kd of 1.4 μM (see FIG. 4g ).Similarly, fluorescence resonance energy transfer (FRET) analysisdemonstrated that CeMMEC1 displaced a tetra-acetylated H4 peptide withgood efficacy from its TAF1 binding site (data not shown). It has beenshown that some kinase inhibitors can behave as bromodomain inhibitors(Ciceri et al., 2014). In order to assess the specificity of CeMMEC1,the inventors tested binding of the compound to the active sites of 97representative kinases profile. None of these kinases were inhibited bymore than 60% at a concentration of 10 μM CeMMEC1, indicatingbromodomain-specificity of the compound (see FIG. 8d ).

Molecular docking was then used to generate hypotheses on the bindingmode of CeMMEC1 and CeMMEC2. CeMMEC2 is a triazolopyridazine and ispredicted to bind to BRD4 similarly to other relatedtriazolophthalazines (Fedorov et al., 2014) (see FIG. 4h ). The triazolenitrogen is predicted to form a hydrogen bond to a conserved asparaginedeep in the peptide binding pocket of BRD4, thereby acting as anacetyllysine mimetic. CeMMEC1 is an N-methylisoquinolinone derivative.Based on the binding of N-methylquinolinone to the bromodomain of ATAD2(Chaikuad et al., 2014), CeMMEC1 can be modeled into the TAF1 pocket(see FIG. 4i ). Its lactam carbonyl is predicted to form a hydrogen bondwith N1604 and with Y1561 through a conserved water molecule. In orderto test this binding mode, the inventors generated a panel of 29 CeMMEC1analogs (see FIG. 12). These compounds were tested for their capabilityto activate RFP in REDS3 cells, and for binding to the bromodomains ofBRD4 (1), BRD4 (2), BRD9, CREBBP, EP300 and TAF1 (2) (see FIG. 8e ).Overall, the data are consistent with the molecular model of CeMMEC1binding, as substitutions on the dihydrobenzodioxin moiety are generallytolerated, and most of the isoquinolinones retain some binding to CREBBPand TAF1. Excitingly, two of the analogs tested, compounds 29 and 30,lost all affinity to CREBBP while retaining TAF1 activity. In contrast,compounds 32 and 35 can serve as negative controls, as they boundneither of the tested bromodomain proteins nor did they induce RFPexpression in REDS3 cells. Both TAF1 specific compounds are structuralisomers of the predicted active-site binding isoquinolinone. In compound29, the isoquinolinone is changed to a quinolinone, whereas in compound30 the attachment point and orientation of the central amide arealtered. The inventors therefore modelled the binding of the specificcompounds to TAF1 (2) and BRD4 (1). Compound 30 was docked into bothproteins, but the dihydrobenzodioxin occupies drastically differentspaces in the binding pockets caused by the different interactions withW1547 in TAF1 and L92 in BRD4 (see FIG. 8f ). The different binding modein TAF1 enables the ligand to form a hydrogen bond with N1554 (see FIG.8g ), likely explaining the specificity. For compound 29, no convincingdocking pose was obtained for BRD4 due to clashes with residues L92 andW81 (see FIG. 8h ).

Finally, as the CeMMEC1-analog 29 appeared to be a selective TAF1inhibitor, the inventors tested for its ability to inhibit binding ofBRD4 (1), BRD9, CREBBP, EP300 and TAF1 (2) bromodomains at 10 μM toacetylated substrate (see FIG. 8i ). Also among this larger panel,compound 29 showed high selectivity for the TAF1 (2) bromodomain.

TAF1 Synergizes with BRD4 to Mediate Transcriptional Control

The results provided herein showed that the inhibition of TAF1phenocopies BRD4 inhibition, indicating a possible functional linkbetween the two bromodomain proteins. Indeed, downregulation of TAF1 inREDS3 cells increased RFP expression (see FIG. 9a ) and decreased c-MYCexpression (see FIG. 5a ) to levels comparable induced by BRD4downregulation. The inventors tested whether these two bromodomainproteins were able to interact directly. 293T cells were transfectedwith BRD4-FLAG and FLAG pull down performed 48 hours later showed thatTAF1 co-immunoprecipitated with FLAG-BRD4 (see FIG. 9b ), indicating adirect interplay of these two bromodomain containing proteins incontrolling gene expression.

As the results provided herein revealed the role of TAF1 in ensuringBRD4 functionality, the inventors asked whether downregulation of TAF1could sensitize cells to the inhibition of BRD4. KBM7 cells treated withshRNAs targeting TAF1 or control hairpins were incubated with differentconcentrations of (S)-JQ1 and cell viability was measured after 96hours. Downregulation of TAF1 decreased cell viability when (S)-JQ1 wasused at concentrations not able to affect control cells, and furtherimpaired cell number at higher concentrations (see FIG. 5b ). Similarly,the synergism of the new direct BRD4 inhibitor, CeMMEC2, with TAF1downregulation was observed (see FIG. 5c ). Furthermore, the additionalinhibition of TAF1 by CeMMEC1 impaired cell viability in TAF1downregulated cells (see FIG. 9c ), indicating that a strong reductionof TAF1 activity alone can be toxic in these cells.

To further provide evidence for a functional relationship between thesetwo bromodomain containing proteins, the inventors simultaneouslyinhibited TAF1 and BRD4, with CeMMEC1 or the analogs 29 or 30 (see FIG.5d ), which showed comparable induction of RFP expression in REDS3 cells(see FIG. 5e ), and (S)-JQ1 respectively. The combination of thesecompounds in REDS3 cells further boosted RFP expression beyond theincrease in single treatments, indicating cooperation between TAF1 andBRD4 on the remodeling of the RFP locus. The same effect was notachieved when using the TAF1-inactive analogs 32 and 35 (see FIG. 5f ).

As KBM7 cells are not particularly sensitive to BRD4 inhibitors, theinventors wanted to test whether the synergy between BRD4 inhibitors andTAF1 inhibitors was conserved in BRD4-dependent cancers. Therefore, itwas tested whether the combined inhibition of TAF1 and BRD4 could arrestthe proliferation of THP1 and H23 cells, a lung adenocarcinoma cell linealso sensitive to the inhibition of BRD4. It was observed that thecombination of (S)-JQ1 and CeMMEC1 was more efficiently impairing cellviability than the individual treatments (see FIGS. 5g and 9d ). TheBliss independence test (Bliss, 1939) confirmed the synergism betweenthese two treatments and showed that the combination between JQ1S andthe analog 29, the most specific in binding TAF1 (2), was the mosteffective (see FIG. 9e ).

Discussion

Chromatin reporter cell lines have been proposed as models to identifymodulators of position effect variegation and chromatin-targeting smallmolecules (Johnson et al., 2008; Best et al., 2011; Wang et al., 2013;Tchasovnikarova et al., 2015). In contrast to previous approaches, theinventors developed a strategy to map chromatin reactivation focused ona specific regulator, BRD4. They selected clones that integratedreporters in fully repressed genomic regions and specifically activatedthe expression of RFP following BRD4 inhibition. The haploid nature ofthe reporter cell line makes it easily amenable to genetic screens, andits application for the identification of genes in BRD4 functionalpathways will provide further insights into BRD4 biology.

With this reporter cell line validated, the inventors first took achemical genomics approach and identified compounds that functionallyantagonize BRD4. In addition to all known BET inhibitors contained intheir library, the inventors identified 13 small molecules that have notbeen linked to BRD4 biology previously. One of these molecules ispanobinostat, a clinically approved histone deacetylase (HDAC)inhibitor. Out of more than 40 HDAC-targeting compounds tested,Panobinostat is the sole compound inducing RFP expression in REDS3cells, indicating a panobinostat-specific activity. These findingsencourage future efforts to fully characterize Panobinostat and allother validated hit compounds regarding their molecular mechanism andprotein targets.

The inventors focused their efforts on two compounds that phenocopy BRD4inhibitors not only by their ability to activate RFP reporter expressionbut also by repressing c-MYC. One of the hit structures, CeMMEC2, turnedout to be a novel direct BRD4 inhibitor. Interestingly, several reportsexist in the patent literature describing compounds related to CeMMEC2to inhibit BRD4 (WO 2014/191894; WO 2014/076146; US 2014/0135336; WO2014/191896; US 2014/0349990; WO 2012/174487). Moreover, the screenprovided herein has also yielded compounds that do not strongly bindBRD4 but still activate the reporter cell line. For one of thesecompounds, CeMMEC1, the inventors have identified TAF1 as the relevanttarget in their system. TAF1 is the largest component of the TAFsubunits contained in the TFIID core, which is part of thepre-initiation complex (PIC) and serves to recognize the TATA box andcorrectly place RNAPol II for transcription initiation (Lee et al.,2005; Kloet et al., 2012; Kandiah et al., 2014). Thereby, TAF1 plays afundamental role in the assembly of the transcription machinery. Similarto BRD4, TAF1 is essential for the viability of many different celllines (Wang et al., 2015; Blomen et al., 2015), and the two proteinsinteract not only in the regulation of transcription but also physicallyin co-immunoprecipitation experiments. The inventors have shown thatTAF1 knockdown increases sensitivity to BRD4 inhibition, and BRD4inhibitors synergize with TAF1 inhibitors, such as CeMMEC1, to impairviability of BRD4-dependent cell lines.

The specific functions of the bromodomains of TAF1 have remainedelusive; the results provided herein indicate that the secondbromodomain of TAF1 is a relevant target in BRD4 driven cancers. CeMMEC1proves druggability of this domain and allows further development ofisoquinolinones as bromodomain inhibitors (Arrowsmith et al., 2015;Workman et al., 2010; Frye, 2010). More selective analogs such asquinolinone 29 open up the avenue to specifically target TAF1 in cancer.

In summary, the results provided herein successfully validate theapplication of haploid epigenetic reporters to identify functionalpathways and novel chemical structures regulating chromatin organizationand transcriptional control.

The results provided herein furthermore demonstrate that the compoundsof formula (I), including the exemplary compounds of formula (I) shownin FIG. 12, are potent inhibitors of TAF1 and can thus be used for thetherapy of diseases/disorders associated with TAF1, particularly for thetreatment or prevention of cancer.

Example 2

The following further compounds of formula (I) according to theinvention were synthesized by Enamine Ltd. (Kiev, Ukraine) following thescheme below:

A mixture of acid 1 (1.1 mmol), amine 2 (1.0 mmol), EDC (1.1 mmol), andHOBt (1.6 mmol) in DMF (1 ml) was stirred at room temperature for 24hours. Chloroform (6 ml) and water (8 ml) was added, organic layer wasseparated, washed with water (8 ml) twice, dried over Na₂SO₄ andevaporated. The crude residue was purified by reversed phase (C-18)chromatography with gradient elution (methanol-water) to yield pure 3.

All compounds were quality controlled by LC-MS, requiring a minimumpurity of 90%.

The binding of these compounds to TAF1 was tested by DiscoverXCorporation (Fremont, Calif., USA) in a primary screen using theBROMOscan assay, which is a ligand binding site-directed competitionassay that allows to quantitatively measure interactions between testcompounds and bromodomains (see Fabian et al., 2005 for an explanationof the principle of this assay), according to the following protocol:

Bromodomain assays: T7 phage strains displaying bromodomains were grownin parallel in 24-well blocks in an E. coli host derived from the BL21strain. E. coli were grown to log-phase and infected with T7 phage froma frozen stock (multiplicity of infection=0.4) and incubated withshaking at 32° C. until lysis (90-150 minutes). The lysates werecentrifuged (5,000×g) and filtered (0.2 μm) to remove cell debris.Streptavidin-coated magnetic beads were treated with biotinylated smallmolecule or acetylated peptide ligands for 30 minutes at roomtemperature to generate affinity resins for bromodomain assays. Theliganded beads were blocked with excess biotin and washed with blockingbuffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to removeunbound ligand and to reduce non-specific phage binding. Bindingreactions were assembled by combining bromodomains, liganded affinitybeads, and test compounds in 1x binding buffer (16% SeaBlock, 0.32x PBS,0.02% BSA, 0.04% Tween 20, 0.004% sodium azide, 7.9 mM DTT). Testcompounds were prepared as 1000X stocks in 100% DMSO and subsequentlydiluted 1:25 in monoethylene glycol (MEG). The compounds were thendiluted directly into the assays such that the final concentrations ofDMSO and MEG were 0.1% and 2.4%, respectively. All reactions wereperformed in polypropylene 384-well plates in a final volume of 0.02 ml.The assay plates were incubated at room temperature with shaking for 1hour and the affinity beads were washed with wash buffer (1x PBS, 0.05%Tween 20). The beads were then re-suspended in elution buffer (1x PBS,0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated atroom temperature with shaking for 30 minutes. The bromodomainconcentration in the eluates was measured by qPCR.

The compounds were tested at a concentration of 1 μM, and the %inhibition was determined as follows:

${\% \mspace{14mu} {inhibition}} = {100 - {\left( \frac{{{test}\mspace{14mu} {compound}\mspace{14mu} {signal}} - {{positive}\mspace{14mu} {control}\mspace{14mu} {signal}}}{{{negative}\mspace{14mu} {control}\mspace{14mu} {signal}} - {{positive}\mspace{14mu} {control}\mspace{14mu} {signal}}} \right) \times 100}}$

negative control=DMSO (0% inhibition)positive control=30 μM BI2536 (100% inhibition)

The % inhibition data for TAF1(BD2) thus obtained are summarized in thetable further below.

In addition, the inhibitor binding constants (Kd values) of thecompounds 29, 30, 4-1 and 4-26 for TAF1(BD2) were subsequentlydetermined by DiscoverX Corporation (Fremont, Calif., USA) using theBROMOscan assay according to the following protocol:

Bromodomain assays: T7 phage strains displaying bromodomains were grownin parallel in 24-well blocks in an E. coli host derived from the BL21strain. E. coli were grown to log-phase and infected with T7 phage froma frozen stock (multiplicity of infection=0.4) and incubated withshaking at 32° C. until lysis (90-150 minutes). The lysates werecentrifuged (5,000×g) and filtered (0.2 μm) to remove cell debris.Streptavidin-coated magnetic beads were treated with biotinylated smallmolecule or acetylated peptide ligands for 30 minutes at roomtemperature to generate affinity resins for bromodomain assays. Theliganded beads were blocked with excess biotin and washed with blockingbuffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to removeunbound ligand and to reduce non-specific phage binding. Bindingreactions were assembled by combining bromodomains, liganded affinitybeads, and test compounds in 1x binding buffer (17% SeaBlock, 0.33x PBS,0.04% Tween 20, 0.02% BSA, 0.004% sodium azide, 7.4 mM DTT). Testcompounds were prepared as 1000X stocks in 100% DMSO. Kds weredetermined using an 11-point 3-fold compound dilution series with oneDMSO control point. All compounds for Kd measurements are distributed byacoustic transfer (non-contact dispensing) in 100% DMSO. The compoundswere then diluted directly into the assays such that the finalconcentration of DMSO was 0.09%. All reactions performed inpolypropylene 384-well plates. Each was a final volume of 0.02 ml. Theassay plates were incubated at room temperature with shaking for 1 hourand the affinity beads were washed with wash buffer (1x PBS, 0.05% Tween20). The beads were then re-suspended in elution buffer (1×PBS, 0.05%Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at roomtemperature with shaking for 30 minutes. The bromodomain concentrationin the eluates was measured by qPCR.

Compound handling: An 11-point 3-fold serial dilution of each testcompound was prepared in 100% DMSO at 1000x final test concentration.All compounds for Kd measurements are distributed by acoustic transfer(non-contact dispensing) in 100% DMSO. The compounds were then diluteddirectly into the assays such that the final concentration of DMSO was0.09%. Most Kds were determined using a compound topconcentration=10,000 nM. If the initial Kd determined was <0.169 nM (thelowest concentration tested), the measurement was repeated with a serialdilution starting at a lower top concentration.

Binding constants (Kds) were calculated with a standard dose-responsecurve using the Hill equation:

${Response} = {{Background} + \frac{{Signal} - {Background}}{1 + \left( {{Kd}^{{Hill}\mspace{14mu} {Slope}}\text{/}{Dose}^{{Hill}\mspace{14mu} {Slope}}} \right)}}$

The Hill Slope was set to −1. Curves were fitted using a non-linearleast square fit with the Levenberg-Marquardt algorithm.

The results thus obtained are reported in the following table as IC₅₀values [μM] for TAF1(BD2).

TAF1_BD2 TAF1_BD2 Compound (% inhibition at 1 μM) (IC50 μM) 29 3.100 304.000 4-1 89 0.250 4-2 9 4-3 20 4-4 48 4-10 3 4-13 3 4-14 20 4-16 344-17 11 4-24 58 4-25 4 4-26 82 0.056 4-28 50 4-29 32 4-31 34 4-32 104-33 11

These results further confirm that the compounds of formula (I)according to the present invention are effective in inhibiting TAF1 andcan thus be used for the therapy of diseases/disorders associated withTAF1, particularly for the treatment or prevention of cancer.

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1. A method of treating cancer, the method comprising administering acompound of the following formula (I) or a pharmaceutically acceptablesalt, solvate or prodrug thereof to a subject in need thereof:

wherein: ring B is a group having the following structure:

one of the ring atoms X₂ and X₃ is N(R^(X1)), and the other one of saidring atoms X₂ and X₃ is C(═O); the ring atom X₁ is selected from thegroup consisting of N(R^(X1)), C(R) and C(═O), and the ring atoms X₄ andX₅ are each independently selected from the group consisting ofN(R^(X1)), C(R^(X3)) and C(═O); wherein at least one of said ring atomsX₁, X₄, and X₅ is different from N(R^(X1)) and C(═O); and furtherwherein if X₃ and X₅ are C(═O), X₄ is N(R^(X1)), and X₁ is C(R), then X₂is N(H); each

is independently a single bond or a double bond, wherein at least one ofany two adjacent bonds

is a single bond; each R^(X1) is independently selected from the groupconsisting of hydrogen, C₁₋₅ alkyl, —CO(C₁₋₅ alkyl), —(C₀₋₃alkylene)-aryl, and heteroaryl, wherein the aryl comprised in said—(C₀₋₃ alkylene)-aryl and said heteroaryl are each optionallysubstituted with one or more groups R^(X11), R^(X2) is selected from thegroup consisting of hydrogen, C₁₋₅alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl,—(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅;haloalkyl), —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃,—(C₀₋₃ alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃alkylene)-CO—O—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl); the twogroups R^(X3) are either mutually linked to form, together with the ringcarbon atoms that they are attached to, a 5- or 6-membered cyclyl groupwhich is optionally substituted with one or more groups R^(X31), or thetwo groups R are each independently selected from the group consistingof hydrogen, C₁₋₅alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —OH, —O(C₁₋₅ alkyl),—O(C₁₋₅ alkylene)-OH, —O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —SH,—S(C₁₋₅alkyl), —NH₂, —NH(C₁₋₅alkyl), —N(C₁₋₅ alkyl)(C₁₋₅ alkyl),halogen, C₁₋₅ haloalkyl, —O—(C₁₋₅ haloalkyl), —CF₃, —CN, —NO₂, —CHO,—CO—(C₁₋₅alkyl), —COOH, —CO—O—(C₁₋₅ alkyl), —O—CO—(C₁₋₅alkyl), —CO—NH₂,—CO—NH(C₁₋₅alkyl), —CO—N(C₁₋₅alkyl)(C₁₋₅ alkyl), —NH—CO—(C₁₋₅alkyl),—N(C₁₋₅alkyl)-CO—(C₁₋₅ alkyl), —SO₂—NH₂, —SO₂—NH(C₁₋₅ alkyl),—SO₂—N(C₁₋₅alkyl)(C₁₋₅ alkyl), —NH—SO₂—(C₁₋₅alkyl), and —N(C₁₋₅alkyl)-SO₂—(C₁₋₅ alkyl); each R^(X11) is independently selected from thegroup consisting of C₁₋₅alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —(C₀₋₃alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O(C₁₋₅alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH₂,—(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)(C₁₋₅alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅haloalkyl),—(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃, —(C₀₋₃alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅alkyl)(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl); eachR^(X31) is independently selected from the group consisting ofC₁₋₅alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅ haloalkyl), —(C₀₋₃alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃, —(C₀₋₃ alkylene)-CN,—(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃ alkylene)-CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃ alkylene)-CO—O—(C₁₋₅ alkyl),—(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—NH₂, —(C₀₋₃alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SO₂—NH₂, —(C₀₋₃alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅ alkyl), and —(C₀₋₃alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl); ring B is attached to theremainder of the compound of formula (I) via the ring carbon atom thatis marked with an asterisk (*) or, if X₄ and X₅ are each C(R^(X3)) andthe two groups R^(X3) are mutually linked to form, together with thering carbon atoms that they are attached to, a 5- or 6-membered cyclylgroup which is optionally substituted with one or more groups R^(X31),then ring B may also be attached to the remainder of the compound offormula (I) via any ring carbon atom of said 5- or 6-membered cyclylgroup; ring A is aryl or heteroaryl, wherein said aryl and saidheteroaryl are each optionally substituted with one or more groupsR^(A), and wherein said heteroaryl is selected from the group consistingof 1,4-benzodioxanyl, benzoxanyl, 1,3-benzodioxolanyl, benzoxolanyl, and1,5-benzodioxepanyl; each R^(A) is independently selected from the groupconsisting of C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —(C₀₋₃alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O(C₁₋₅alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH₂,—(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl), (C₁₋₅alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅ haloalkyl),—(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃, —(C₀₋₃alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-cycloalkyl, —(C₀₋₃ alkylene)-O-cycloalkyl, —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-cycloalkyl, —(C₀₋₃alkylene)-heterocycloalkyl, —(C₀₋₃ alkylene)-O-heterocycloalkyl, and—(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-heterocycloalkyl; L is selected fromthe group consisting of —CO—N(R^(L1))—, —N(R^(L1))—CO—, —CO—O—, —O—CO—,—C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—, —C(═S)—N(R^(L1))—,—N(R^(L1))—C(═S)—, —N(R^(L1))—CO—N(R^(L1))—, —O—CO—N(R^(L1))—, —N(R^(L1))—CO—O—, —N(R^(L1))—C(═N—R^(L2))—N(R^(L1))—,—O—C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—O—,—S—C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—S—,—N(R^(L1))—C(═S)—N(R^(L1))—, —O—C(═S)—N(R^(L1))—, —N(R^(L1))—C(═S)—O—,—S—CO—N(R^(L1))—, and —N(R^(L1))—CO—S—; each R^(L1) is independentlyselected from the group consisting of hydrogen and C₁₋₅ alkyl; eachR^(L2) is independently selected from the group consisting of hydrogen,C₁₋₅ alkyl, —CN, and —NO₂; n is 0 or 1; and m is 0 or
 1. 2.-3.(canceled)
 4. The method of claim 1, wherein ring B is attached to theremainder of the compound of formula (I) via the ring carbon atom thatis marked with an asterisk.
 5. (canceled)
 6. The method of claim 1,wherein X₄ and X₅ are each C(R^(X3)) and the two groups R^(X3) aremutually linked to form, together with the ring carbon atoms that theyare attached to, a 5- or 6-membered cycloalkyl group, a 5- or 6-memberedcycloalkenyl group, or a phenyl group, wherein said cycloalkyl group,said cycloalkenyl group and said phenyl group are each optionallysubstituted with one or more groups R^(X31).
 7. (canceled)
 8. The methodof claim 1, wherein the compound of formula (I) has the followingstructure:


9. The method of claim 1, wherein X₂ is C(═O), and X₃ is N(R^(X1)). 10.(canceled)
 11. The method of claim 1, wherein ring A is selected fromthe group consisting of phenyl, 1,4-benzodioxanyl, benzoxanyl,1,3-benzodioxolanyl, benzoxolanyl, and 1,5-benzodioxepanyl, wherein saidphenyl, said 1,4-benzodioxanyl, said benzoxanyl, said1,3-benzodioxolanyl, said benzoxolanyl, and said 1,5-benzodioxepanyl areeach optionally substituted with one or more groups R^(A). 12.(canceled)
 13. The method of claim 1, wherein L is —CO—N(R^(L1))— or—N(R′)—CO—.
 14. The method of claim 1, wherein the moiety—(CH₂)_(n)-L-(CH₂)_(m)— is —(CH₂)_(n)—N(R^(L1))—CO—(CH₂)_(m)—, n is 0,and m is
 0. 15. The method of claim 1, wherein said compound is acompound of any one of the following formulae:

or a pharmaceutically acceptable salt, solvate or prodrug thereof. 16.(canceled)
 17. The method of claim 1, wherein said cancer is selectedfrom the group consisting of prostate carcinoma, breast cancer, acutemyeloid leukemia, multiple myeloma, glioblastoma, and NUT midlinecarcinoma.
 18. A pharmaceutical composition comprising a compound of thefollowing formula (I):

wherein: ring B is a group having the following structure:

one of the ring atoms X₂ and X₃ is N(R^(X1)), and the other one of saidring atoms X₂ and X₃ is C(═O); the ring atom X₁ is C(R)(²); the ringatoms X₄ and X₅ are each C(R), and the two groups R are mutually linkedto form, together with the ring carbon atoms that they are attached to,a 5- or 6-membered cyclyl group which is optionally substituted with oneor more groups R^(X31); R^(X1) is selected from the group consisting ofhydrogen, C₁₋₅ alkyl, —CO(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-aryl, andheteroaryl, wherein the aryl comprised in said —(C₀₋₃ alkylene)-aryl andsaid heteroaryl are each optionally substituted with one or more groupsR^(X11); R^(X2) is selected from the group consisting of hydrogen,C₁₋₅alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅ haloalkyl), —(C₀₋₃alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃, —(C₀₋₃ alkylene)-CN,—(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃ alkylene)-CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃ alkylene)-CO—O—(C₁₋₅ alkyl),—(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—NH₂, —(C₀₋₃alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SO₂—NH₂, —(C₀₋₃alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅ alkyl), and —(C₀₋₃alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl); each R^(X11) is independentlyselected from the group consisting of C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅haloalkyl), —(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃,—(C₀₋₃ alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), and —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl); eachR^(X31) is independently selected from the group consisting of C₁₋₅alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —(C₀₋₃ alkylene)-OH, —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅alkyl), —(C₀₋₃ alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃alkylene)-NH₂, —(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅haloalkyl), —(C₀₋₃ alkylene)-CF₃, —(C₀₋₃ alkylene)-CN, —(C₀₋₃alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃ alkylene)-CO—(C₁₋₅ alkyl),—(C₀₋₃ alkylene)-COOH, —(C₀₋₃ alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—NH₂, —(C₀₋₃alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SO₂—NH₂, —(C₀₋₃alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅ alkyl), and —(C₀₋₃alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl); and ring B is attached to theremainder of the compound of formula (I) via the ring carbon atom thatis marked with an asterisk (*), or ring B is attached to the remainderof the compound of formula (I) via any ring carbon atom of the 5- or6-membered cyclyl group that is formed from the two mutually linkedgroups R^(X3); ring A is aryl or heteroaryl, wherein said aryl and saidheteroaryl are each optionally substituted with one or more groupsR^(A), and wherein said heteroaryl is selected from the group consistingof 1,4-benzodioxanyl, benzoxanyl, 1,3-benzodioxolanyl, benzoxolanyl, and1,5-benzodioxepanyl; each R^(A) is independently selected from the groupconsisting of C₁₋₅ alkyl, C₂₋₅ alkenyl, C₂₋₅ alkynyl, —(C₀₋₃alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O(C₁₋₅alkylene)-OH, —(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-O(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SH, —(C₀₋₃ alkylene)-S(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH₂,—(C₀₋₃ alkylene)-NH(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)(C₁₋₅alkyl), —(C₀₋₃ alkylene)-halogen, —(C₀₋₃ alkylene)-(C₁₋₅ haloalkyl),—(C₀₋₃ alkylene)-O—(C₁₋₅ haloalkyl), —(C₀₋₃ alkylene)-CF₃, —(C₀₋₃alkylene)-CN, —(C₀₋₃ alkylene)-NO₂, —(C₀₋₃ alkylene)-CHO, —(C₀₋₃alkylene)-CO—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-COOH, —(C₀₋₃alkylene)-CO—O—(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-O—CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—NH₂, —(C₀₋₃ alkylene)-CO—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-CO—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—CO—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-CO—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—NH₂, —(C₀₋₃ alkylene)-SO₂—NH(C₁₋₅ alkyl), —(C₀₋₃alkylene)-SO₂—N(C₁₋₅ alkyl)(C₁₋₅ alkyl), —(C₀₋₃ alkylene)-NH—SO₂—(C₁₋₅alkyl), —(C₀₋₃ alkylene)-N(C₁₋₅ alkyl)-SO₂—(C₁₋₅ alkyl), —(C₀₋₃alkylene)-cycloalkyl, —(C₀₋₃ alkylene)-O-cycloalkyl, —(C₀₋₃alkylene)-O(C₁₋₅ alkylene)-cycloalkyl, —(C₀₋₃alkylene)-heterocycloalkyl, —(C₀₋₃ alkylene)-O-heterocycloalkyl, and—(C₀₋₃ alkylene)-O(C₁₋₅ alkylene)-heterocycloalkyl; L is selected fromthe group consisting of —CO—N(R^(L1))—, —N(R^(L1))—CO—, —CO—O—, —O—CO—,—C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—, —C(═S)—N(R^(L1))—,—N(R^(L1))—C(═S)—, —N(R^(L1))—CO—N(R^(L1))—, —O—CO—N(R^(L1))—, —N(R^(L1))—CO—O—, —N(R^(L1))—C(═N—R^(L2))—N—R^(L1))—,—O—C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—O—,—S—C(═N—R^(L2))—N(R^(L1))—, —N(R^(L1))—C(═N—R^(L2))—S—,—N(R^(L1))—C(═S)—N(R^(L1))—, —O—C(═S)—N(R^(L1))—, —N(R^(L1))—C(═S)—O—,—S—CO—N(R^(L1))—, and —N(R^(L1))—CO—S—; each R^(L1) is independentlyselected from the group consisting of hydrogen and C₁₋₅ alkyl; eachR^(L2) is independently selected from the group consisting of hydrogen,C₁₋₅ alkyl, —CN, and —NO₂; n is 0 or 1; and m is 0 or 1; or apharmaceutically acceptable salt, solvate or prodrug thereof; and apharmaceutically acceptable excipient.
 19. The compound pharmaceuticalcomposition of claim 18, wherein ring B is attached to the remainder ofthe compound of formula (I) via the ring carbon atom that is marked withan asterisk.
 20. (canceled)
 21. The pharmaceutical composition of claim18, wherein the two groups R are mutually linked to form, together withthe ring carbon atoms that they are attached to, a 5- or 6-memberedcycloalkyl group, a 5- or 6-membered cycloalkenyl group, or a phenylgroup, wherein said cycloalkyl group, said cycloalkenyl group and saidphenyl group are each optionally substituted with one or more groupsR^(X31). 22.-23. (canceled)
 24. The pharmaceutical composition of claim18, wherein the compound of formula (I) has the following structure:


25. The pharmaceutical composition of claim 18, wherein X₂ is C(═O), andX₃ is N(R^(X1)).
 26. (canceled)
 27. The pharmaceutical composition ofclaim 18, wherein ring A is selected from the group consisting ofphenyl, 1,4-benzodioxanyl, benzoxanyl, 1,3-benzodioxolanyl,benzoxolanyl, and 1,5-benzodioxepanyl, wherein said phenyl, said1,4-benzodioxanyl, said benzoxanyl, said 1,3-benzodioxolanyl, saidbenzoxolanyl, and said 1,5-benzodioxepanyl are each optionallysubstituted with one or more groups R^(A).
 28. (canceled)
 29. Thepharmaceutical composition of claim 18, wherein L is —CO—N(R^(L1))— or—N(R′)—CO—.
 30. (canceled)
 31. The pharmaceutical composition of claim18, wherein said compound is a compound of any one of the followingformulae:

or a pharmaceutically acceptable salt, solvate or prodrug thereof. 32.(canceled)
 33. The method of claim 1, wherein the method comprisesadministering said compound in combination with a BRD4 inhibitor. 34.The method of claim 33, wherein said BRD4 inhibitor is selected fromCeMMEC2, (S)-JQ1, I-BET 151, I-BET 762, PF-1, bromosporine, OTX-015,TEN-010, CPI-203, CPI-0610, RVX-208, BI2536, TG101348, LY294002, or apharmaceutically acceptable salt, solvate or prodrug of any one of theseagents. 35.-37. (canceled)
 38. A compound having any one of thefollowing formulae:

or a pharmaceutically acceptable salt, solvate or prodrug thereof. 39.(canceled)
 40. A method of treating cancer, the method comprisingadministering a TAF1 inhibitor in combination with a BRD4 inhibitor to asubject in need thereof. 41.-44. (canceled)
 45. The method of claim 1,wherein the subject is a human.